Isolation and Identification of Glycosaminoglycans

ABSTRACT

The isolation and identification of glycosaminoglycans capable of binding to proteins having a heparin-binding domain is disclosed, as well as the use of the glycosaminoglycans isolated in the growth and/or development of tissue.

FIELD OF THE INVENTION

The present invention relates to the isolation and identification ofglycosaminoglycans capable of binding to proteins having aheparin-binding domain, as well as to the use of the glycosaminoglycansisolated in the growth and/or development of tissue.

BACKGROUND TO THE INVENTION

Glycosaminoglycans (GAGs) are complex carbohydrate macromoleculesresponsible for performing and regulating a vast number of essentialcellular functions.

GAGs have been implicated in the modulation or mediation of manysignalling systems in concert with the many hundreds of knownheparin-binding growth and adhesive factors. It is contemplated that theassociation of growth factors with GAGs modulates their variousactivities with a diverse range of actions, such as lengthening theirhalf-lives by protecting them from proteolytic degradation, modulatinglocalisation of these cytokines at the cell surface, mediating molecularinteractions and stabilising ligand-receptor complexes.

There are an ever increasing number of identified heparin-binding growthfactors, adding to the hundreds already known, most of which werepurified by heparin affinity chromatography. They include the extensivefibroblast growth factor (FGF) family, the PDGFs and the pleiotropinsthrough to the TGF-β superfamily of cytokines. This latter family offactors encompasses the osteo-inductive bone morphogenetic protein (BMP)subfamily, so named for their ability to induce ectopic bone formation.

The nature and effect of the interaction of GAGs and growth factorsremains unclear. Although the interaction between FGF2 and particularsaccharide sequences found within heparin has been shown to be of highaffinity, it remains generally unclear whether the association betweenother growth factors and heparans involves a high affinity or specificbinding interaction between an amino acid sequence epitope on theprotein growth factor and a saccharide sequence embedded in the GAG, orwhether the association is mediated by lower affinity, non-specificinteractions between the GAG and protein growth factor.

If interactions between GAGs and proteins resident in, or secreted into,the extracellular matrix are specific, the binding partners need to beidentified in order to unravel the interactions and understand how theseinteractions may be used or modulated to provide new treatments.

A major question that arises is, therefore, whether there are saccharidesequences embedded in the chains of GAG molecules that match primaryamino acid sequences within the polypeptide backbone of growth factorsso controlling their association, and so bioactivity, with absolute, orat least relative, specificity.

SUMMARY OF THE INVENTION

We have devised a method to answer this question which involvesenriching for glycosaminoglycan molecules that exhibit binding toparticular polypeptides having a heparin-binding domain. Isolated GAGmixtures and/or molecules can then be identified and tested for theirability to modulate the growth and differentiation of cells and tissueexpressing a protein containing the heparin-binding domain. For thefirst time, this enables the controlled analysis of the effect ofparticular GAG saccharide sequences on the growth and differentiation ofcells and tissue, both in vitro and in vivo.

Accordingly, in a first aspect of the present invention a method ofisolating glycosaminoglycans capable of binding to proteins havingheparin/heparan-binding domains is provided, the method comprising:

-   -   (i) providing a solid support having polypeptide molecules        adhered to the support, wherein the polypeptide comprises a        heparin-binding domain;    -   (ii) contacting the polypeptide molecules with a mixture        comprising glycosaminoglycans such that        polypeptide-glycosaminoglycan complexes are allowed to form;    -   (iii) partitioning polypeptide-glycosaminoglycan complexes from        the remainder of the mixture;    -   (iv) dissociating glycosaminoglycans from the        polypeptide-glycosaminoglycan complexes;    -   (v) collecting the dissociated glycosaminoglycans.

In another aspect of the present invention isolated glycosaminoglycansare identified by their ability to modulate the growth ordifferentiation of cells or tissues. A method of identifyingglycosaminoglycans capable of stimulating or inhibiting the growthand/or differentiation of cells and/or tissues is provided, the methodcomprising:

-   -   (i) providing a solid support having polypeptide molecules        adhered to the support, wherein the polypeptide comprises a        heparin-binding domain;    -   (ii) contacting the polypeptide molecules with a mixture        comprising glycosaminoglycans such that        polypeptide-glycosaminoglycan complexes are allowed to form;    -   (iii) partitioning polypeptide-glycosaminoglycan complexes from        the remainder of the mixture;    -   (iv) dissociating glycosaminoglycans from the        polypeptide-glycosaminoglycan complexes;    -   (v) collecting the dissociated glycosaminoglycans;    -   (vi) adding the collected glycosaminoglycans to cells or tissues        in which a protein containing the amino acid sequence of the        heparin-binding domain is present;    -   (vii) measuring one or more of: proliferation of the cells,        differentiation of the cells, expression of one or more protein        markers.

In embodiments of the present invention the mixture comprising GAGs maycontain synthetic glycosaminoglycans. However, in preferred embodimentsGAGs obtained from cells or tissues are used. For example, the mixturemay contain extracellular matrix wherein the extracellular matrixmaterial is obtained by scraping live tissue in situ (i.e. directly fromthe tissue in the body of the human or animal from which it is obtained)or by scraping tissue (live or dead) that has been extracted from thebody of the human or animal. Alternatively, the extracellular matrixmaterial may be obtained from cells grown in culture. The extracellularmatrix material may be obtained from connective tissue or connectivetissue cells, e.g. bone, cartilage, muscle, fat, ligament or tendon.

The GAG component may be extracted from a tissue or cell sample orextract by a series of routine separation steps (e.g. anion exchangechromatography), well known to those of skill in the art.

GAG mixtures may contain a mixture of different types ofglycosaminoglycan, which may include dextran sulphates, chondroitinsulphates and heparan sulphates. In preferred embodiments the GAGmixture contacted with the solid support has been enriched for one ofthese types of glycosaminoglycan, most preferably for heparan sulphate.A heparan sulphate-, chondroitin sulphate- or dextran sulphate-enrichedGAG fraction may be obtained by performing column chromatography on theGAG mixture, e.g. weak, medium or strong anion exchange chromatography,as well as strong high pressure liquid chromatography (SAX-HPLC), withselection of the appropriate fraction.

The collected GAGs may be subjected to further analysis in order toidentify the GAG, e.g. determine GAG composition or sequence, ordetermine structural characteristics of the GAG. GAG structure istypically highly complex, and, taking account of currently availableanalytical techniques, exact determinations of GAG sequence structureare not possible in most cases.

However, the collected GAG molecules may be subjected to partial orcomplete saccharide digestion (e.g. chemically by nitrous acid orenzymatically with lyases such as heparinase III) to yield saccharidefragments that are both characteristic and diagnostic of the GAG. Inparticular, digestion to yield disaccharides (or tetrasaccharides) maybe used to measure the percentage of each disaccharide obtained whichwill provide a characteristic disaccharide “fingerprint” of the GAG.

The pattern of sulphation of the GAG can also be determined and used todetermine GAG structure. For example, for heparan sulphate the patternof sulphation at amino sugars and at the C2, C3 and C6 positions may beused to characterise the heparan sulphate.

Disaccharide analysis, tetrasaccharide analysis and analysis ofsuphation can be used in conjunction with other analytical techniquessuch as HPLC, mass spectrometry and NMR which can each provide uniquespectra for the GAG. In combination, these techniques may provide adefinitive structural characterisation of the GAG.

A high affinity binding interaction between the GAG and heparin-bindingdomain indicates that the GAG will contain a specific saccharidesequence that contributes to the high affinity binding interaction. Afurther step may comprise determination of the complete or partialsaccharide sequence of the GAG, or the key portion of the GAG, involvedin the binding interaction.

In one embodiment, GAG-polypeptide complexes may be subjected totreatment with an agent that lyses glycosaminoglycan chains, e.g. alyase. Lyase treatment may cleave portions of the bound GAG that are nottaking part in the binding interaction with the polypeptide. Portions ofthe GAG that are taking part in the binding interaction with thepolypeptide may be protected from lyase action. After removal of thelyase, e.g. following a washing step, the GAG molecule that remainsbound to the polypeptide represents the specific binding partner (“GAGligand”) of the polypeptide. Owing to the lower complexity of shorterGAG molecules, following dissociation and collection of the GAG ligand,a higher degree of structural characterisation of the GAG ligand can beexpected. For example, the combination of any of the saccharide sequence(i.e. the primary (linear) sequence of monosaccharides contained in theGAG ligand), sulphation pattern, disaccharide and/or tetrasaccharidedigestion analysis, NMR spectra, mass spectrometry spectra and HPLCspectra may provide a high level of structural characterisation of theGAG ligand.

In one aspect of the present invention a GAG is provided having highbinding affinity for BMP2. More preferably the GAG is a heparan sulphate(HS). The HS was isolated from a GAG mixture obtained from theextracellular matrix of osteoblasts by following the methodology of thepresent invention in which a polypeptide comprising the heparin-bindingdomain of BMP2 (SEQ ID NO:1) was attached to a solid support andGAG-polypeptide complexes were allowed to form. Dissociation of the GAGcomponent from the GAG-polypeptide complexes led to isolation of aunique HS herein called “HS/BMP2”.

Accordingly, in one aspect of the present invention HS/BMP2 is provided.HS/BMP2 may be provided in isolated or purified form. In another aspectculture media comprising HS/BMP2 is provided.

In yet another aspect of the present invention a pharmaceuticalcomposition or medicament comprising HS/BMP2 is provided, optionally incombination with a pharmaceutically acceptable carrier, adjuvant ordiluent. In some embodiments pharmaceutical compositions or medicamentsmay further comprise BMP2 protein. Pharmaceutical compositions ormedicaments comprising HS/BMP2 are provided for use in the prevention ortreatment of injury or disease. The use of HS/BMP2 in the manufacture ofa medicament for the prevention or treatment of injury or disease isalso provided.

In a further aspect of the present invention, a method of preventing ortreating injury or disease in a patient in need of treatment thereof isprovided, the method comprising administering an effective amount ofHS/BMP2 to the patient. The administered HS/BMP2 may be formulated in asuitable pharmaceutical composition or medicament and may furthercomprise a pharmaceutically acceptable carrier, adjuvant or diluent.Optionally, the pharmaceutical composition or medicament may alsocomprise BMP2 protein.

In another aspect of the present invention a method of promoting orinhibiting osteogenesis (the formation of bone cells and/or bone tissue)is provided comprising administering HS/BMP2 to bone precursor cells orbone stem cells.

In another aspect of the present invention a method of promoting orinhibiting the formation of cartilage tissue (chondrogenesis) isprovided, comprising administering HS/BMP2 to cartilage precursor cellsor cartilage stem cells.

The methods of stimulating or inhibiting osteogenesis or formation ofcartilage tissue may be conducted in vitro by contacting bone orcartilage precursor or stem cells with HS/BMP2, optionally in thepresence of exogenously added BMP2 protein. The precursor cells or stemcells may be mesenchymal stem cells. Where tissue formation is promoted,the tissue formed may be collected and used for implantation into ahuman or animal patient.

Accordingly, in one aspect of the present invention, connective tissueis provided wherein the connective tissue is obtained by in vitroculture of mesenchymal stem cells in the presence of HS/BMP2 (i.e.exogenous HS/BMP2), and optionally in the presence of BMP2 (i.e.exogenous BMP2). The connective tissue may be bone, cartilage, muscle,fat, ligament or tendon.

The prevention or treatment of disease using HS/BMP2 may involve therepair, regeneration or replacement of tissue, particularly connectivetissue such as bone, cartilage, muscle, fat, ligament or tendon.

In patients having a deterioration of one of these tissues,administration of HS/BMP2 to the site of deterioration may be used tostimulate the growth, proliferation and/or differentiation of tissue atthat site. For example, stimulation of mesenchymal stem cells presentat, or near to, the site of administration may lead, preferably whenBMP2 is also present at the site, to the proliferation anddifferentiation of the mesenchymal stem cells into the appropriateconnective tissue, thereby providing for replacement/regeneration of thedamaged tissue and treatment of the injury.

Alternatively, connective tissue obtained from in vitro culture ofmesenchymal stem cells in contact with HS/BMP2 may be collected andimplanted at the site of injury or disease to replace damaged ordeteriorated tissue. The damaged or deteriorated tissue may optionallyfirst be excised from the site of injury or disease.

In another aspect, a pharmaceutical composition may be providedcontaining stem cells, preferably mesenchymal stem cells, and HS/BMP2.Administration, e.g. injection, of the composition at the site ofinjury, disease or deterioration provides for the regeneration of tissueat the site.

Accordingly, HS/BMP2 is useful in wound healing in vivo, includingtissue repair, regeneration and/or replacement (e.g. healing of scartissue or a broken bone) effected by direct application of HS/BMP2,optionally in combination with BMP2 and/or stem cells, to the patientrequiring treatment. HS/BMP2 is also useful in the in vitro generationof tissue suitable for implantation into a patient in need of tissuerepair, regeneration and/or replacement.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to GAGs, and especially to methods ofenriching mixtures of compounds containing one or more GAGs that bind toa polypeptide corresponding to the heparin-binding domain of a proteinthat binds heparin/heparan (a “heparin-binding factor”). Enrichmentleads to isolation of GAGs, whether as a mixture containing differentGAGs or a population of GAGs that are structurally or functionallyidentical (or substantially identical). The enriched mixture preferablyhas a modulating effect on the heparin-binding factor.

The present invention also relates to mixtures of compounds enrichedwith one or more GAGs which possess a modulating effect on aheparin/heparan-binding factor, and methods of using such mixtures.

The present invention also relates to GAG molecules which potentiate(e.g. agonize) the activity of BMP-2 and hence its ability to stimulatestem cell proliferation and bone formation.

As used herein, the terms ‘enriching’, ‘enrichment’, ‘enriched’, etc.describes a process (or state) whereby the relative composition of amixture is (or has been) altered in such a way that the fraction of thatmixture given by one or more of those entities is increased, while thefraction of that mixture given by one or more different entities isdecreased.

GAGs isolated by enrichment may be pure, i.e. contain substantially onlyone type of GAG, or may continue to be a mixture of different types ofGAG, the mixture having a higher proportion of particular GAGs that bindto the heparin-binding domain relative to the starting mixture.

GAGs identified by the present invention are preferably GAGs thatexhibit a functional effect when contacted with cells or tissue in whicha protein containing the heparin-binding domain is expressed orcontained. The functional effect may be a modulating or potentiatingeffect.

The functional effect may be to promote (stimulate) or inhibit theproliferation of the cells of a certain type or the differentiation ofone cell type into another, or the expression of one or more proteinmarkers. For example, the GAGs may promote cell proliferation, i.e. anincrease in cell number, or promote differentiation of stem cells intospecialised cell types (e.g. mesenchymal stem cells in connectivetissue), promote or inhibit the expression of protein markers indicativeof the multipotency or differentiation state of the cells (e.g. markerssuch as alkaline phosphatase activity, detection of RUNX2, osterix,collagen I, II, IV, VII, X, osteopontin, Osteocalcin, BSPII, SOX9,Aggrecan, ALBP, CCAAT/enhancer binding protein-α (C/EBPα), adipocytelipid binding protein (ALBP), alkaline phosphatise (ALP), bonesialoprotein 2, (BSPII), Collagen2a1 (CoII2a) and SOX9).

As used herein, the term ‘modulating effect’ is understood to mean theeffect that a first entity has on a second entity wherein the secondentity's normal function in another process or processes is modified bythe presence of the first entity. In a preferred embodiment of thepresent invention, the modulating effect may be either agonistic orantagonistic.

The modulating effect may be a potentiating effect. The term‘potentiating effect’ is understood to mean the effect of increasingpotency. In a preferred embodiment of the present invention, the term‘potentiating effect’ refers to the effect that a first entity has on asecond entity, which effect increases the potency of that second entityin another process or processes. In a further preferred embodiment ofthe present invention, the potentiating effect is understood to mean theeffect of isolated GAGs on a heparin-binding factor, wherein the saideffect increases the potency of said heparin-binding factor.

In a preferred embodiment of the present invention, the potentiatingeffect is an increase in bioavailability of the heparin-binding factor.In a preferred embodiment of the present invention, the potentiatingeffect is an increase in bioavailability of BMP2. One method ofmeasuring an increase in bioavailability of the heparin-binding factoris through determining an increase in local concentration of theheparin-binding factor.

In another embodiment of the present invention, the potentiating effectis to protect the heparin-binding factor from degradation. In anespecially preferred embodiment of the present invention, thepotentiating effect is to protect BMP-2 from degradation. One method ofdetermining a decrease in the degradation of the heparin-binding factoris through measuring an increase in the half-life of the heparin-bindingfactor.

In another embodiment of the present invention, the potentiating effectis to sequester heparin-binding factors away from cellular receptors. Inanother embodiment of the present invention, the potentiating effect isto stabilise the ligand-receptor interaction.

The potentiating effect (e.g. modulation of growth or differentiation)may be determined by use of appropriate assays. For example, the effectthat an HS has on the stability of BMP-2 may be determined by ELISA. Theeffect that an HS has on the activity of BMP-2 may be determined bymeasuring the activation/expression of one or more of SMAD 1, 5 or 8, ormeasuring the expression of one or more osteogenic marker genes such asRunx2, alkaline phosphatase, Osterix, Osteocalcin and BSP1, or measuringthe levels of mineralization using staining such as Alizarin Red and vonKossa.

As used herein, the process of ‘contacting’ involves the bringing intoclose physical proximity of two or more discrete entities. The processof ‘contacting’ involves the bringing into close proximity of two ormore discrete entities for a time, and under conditions, sufficient toallow a portion of those two or more discrete entities to interact on amolecular level. Preferably, as used herein, the process of ‘contacting’involves the bringing into close proximity of the mixture of compoundspossessing one or more GAGs and the polypeptide corresponding to theheparin-binding domain of a heparin-binding factor. Examples of‘contacting’ processes include mixing, dissolving, swelling, washing. Inpreferred embodiments ‘contact’ of the GAG mixture and polypeptide issufficient for complexes, which may be covalent but are preferablynon-covalent, to form between GAGs and polypeptides that exhibit highaffinity for each other.

The polypeptide may comprise the full length or near full length primaryamino acid sequence of a selected protein having a heparin-bindingdomain. Due to folding that may occur in longer polypeptides leading topossible masking of the heparin-binding domain from the GAG mixture, itis preferred for the polypeptide to be short. Preferably, thepolypeptide will have an amino acid sequence that includes theheparin-binding domain and optionally including one or more amino acidsat one or each of the N- and C-terminals of the peptides. Theseadditional peptides may enable the addition of linker or attachmentmolecules to the polypeptide that are required to attach the polypeptideto the solid support.

In preferred embodiments in addition to the number of amino acids in theheparin-binding domain the polypeptide contains 1-20, more preferably1-10, still more preferably 1-5 additional amino acids. In someembodiments the amino acid sequence of the heparin-binding domainaccounts for at least 80% of the amino acids of the polypeptide, morepreferably at least 90%, still more preferably at least 95%.

In order to adhere polypeptides to the surface of a solid support thepolypeptides are preferably modified to include a molecular tag, and thesurface of the solid support is modified to incorporate a correspondingmolecular probe having high affinity for the molecular tag, i.e. themolecular tag and probe form a binding pair. In preferred embodimentsthe tag and/or probe is chosen from any one of: an antibody, a cellreceptor, a ligand, biotin, any fragment or derivative of thesestructures, any combination of the foregoing, or any other structurewith which a probe can be designed or configured to bind or otherwiseassociate with specificity. A preferred binding pair suitable for use astag and probe is biotin and avidin.

The polypeptide is preferably derived from a protein of interest. By“derived from” is meant that the polypeptide is chosen, selected orprepared because it contains the amino acid sequence of aheparin-binding domain that is present in a protein of interest. In someembodiments, the amino acid sequence of the heparin-binding domain maybe modified from that appearing in the protein of interest, e.g. toinvestigate the effect of changes in the heparin-binding domain sequenceon GAG binding.

The protein of interest may be any protein that binds heparin, andtherefore has a heparin-binding domain. Preferred proteins include thoseexpressed in the extracellular matrix, in particular in theextracellular matrix of connective tissue (e.g. bone, cartilage, muscle,tendons, ligaments, fat).

Preferred proteins and their heparin-binding domains are set out below:

SEQ Amino acid sequence of ID Protein heparin-binding domain NO. BMP2QAKHKQRKRLKSSCKRHP  1 BMP4 SPKHHSQRARKKNKNCRRH  2 FGF2TYRSRKYTSWYVALKRTGQYKLGSKTGPGQK  3 SHH GKRRHPKKLTPLAYKQ  4 VEGF KCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWS  5 189 FGFR1APYWTSPEKMEKKLHAVPAAKTVK  6 VITRO- RPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRR  7NECTIN PDGF B RVRRPPKGKHRKFKHTH  8 HB-EGF HGKRKKKGKGLGKKRDPCLRKYK  9FGFR3 APYWTRPERMDKKLLAVPAANTVR 10 FIBRO- TLENVSPPRRARV 11 NECTIN LAMININRYVVLPRPVCFEKGMNYTVR 12 N-CAM IWKHKGRDVILKKDVRFI 13

It is understood by those skilled in the art that small variations inthe amino acid sequence of a particular polypeptide may allow theinherent functionality of that portion to be maintained. It is alsounderstood that the substitution of certain amino acid residues within apeptide with other amino acid residues that are isosteric and/orisoelectronic may either maintain or improve certain properties of theunsubstituted peptide. These variations are also encompassed within thescope of the present invention. For example, the amino acid alanine maysometimes be substituted for the amino acid glycine (and vice versa)whilst maintaining one or more of the properties of the peptide. Theterm ‘isosteric’ refers to a spatial similarity between two entities.Two examples of moieties that are isosteric at moderately elevatedtemperatures are the iso-propyl and tert-butyl groups. The term‘isoelectronic’ refers to an electronic similarity between two entities,an example being the case where two entities possess a functionality ofthe same, or similar, pKa.

In embodiments of the present invention, the polypeptide correspondingto the heparin-binding domain may be synthetic or recombinant.

The solid support may be any substrate having a surface to whichmolecules may be attached, directly or indirectly, through eithercovalent or non-covalent bonds. The solid support may include anysubstrate material that is capable of providing physical support for theprobes that are attached to the surface. It may be a matrix support. Thematerial is generally capable of enduring conditions related to theattachment of the probes to the surface and any subsequent treatment,handling, or processing encountered during the performance of an assay.The materials may be naturally occurring, synthetic, or a modificationof a naturally occurring material. The solid support may be a plasticsmaterial (including polymers such as, e.g., poly(vinyl chloride),cyclo-olefin copolymers, polyacrylamide, polyacrylate, polyethylene,polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate,poly(ethylene terephthalate), polytetrafluoroethylene (PTFE or Teflon®),nylon, poly(vinyl butyrate)), etc., either used by themselves or inconjunction with other materials. Additional rigid materials may beconsidered, such as glass, which includes silica and further includes,for example, glass that is available as Bioglass. Other materials thatmay be employed include porous materials, such as, for example,controlled pore glass beads. Any other materials known in the art thatare capable of having one or more functional groups, such as any of anamino, carboxyl, thiol, or hydroxyl functional group, for example,incorporated on its surface, are also contemplated.

Preferred solid supports include columns having a polypeptideimmobilized on a surface of the column. The surface may be a wall of thecolumn, and/or may be provided by beads packed into the central space ofthe column.

The polypeptide may be immobilised on the solid support. Examples ofmethods of immobilisation encompassed within the scope of the presentinvention include: adsorption, covalent binding, entrapment and membraneconfinement. In a preferred embodiment of the present invention theinteraction between the polypeptide and the matrix is substantiallypermanent. In a further preferred embodiment of the present invention,the interaction between the peptide and the matrix is suitably inert toion-exchange chromatography. In a preferred embodiment, the polypeptideis attached to the surface of the solid support. It is understood that aperson skilled in the art would have a large array of options to choosefrom to chemically and/or physically attach two entities to each other.These options are all encompassed within the scope of the presentinvention. In a preferred embodiment of the present invention, thepolypeptide is adsorbed to a solid support through the interaction ofbiotin with streptavidin. In a representative example of this particularembodiment, a molecule of biotin is bonded covalently to thepolypeptide, whereupon the biotin-polypeptide conjugate binds tostreptavidin, which in turn has been covalently bonded to a solidsupport. In another embodiment of the present invention, a spacer orlinker moiety may be used to connect the molecule of biotin with thepolypeptide, and/or the streptavidin with the matrix.

By contacting the GAG mixture with the solid support GAG-polypeptidecomplexes are allowed to form. These are partitioned from the remainderof the mixture by removing the remainder of the mixture from the solidsupport, e.g. by washing the solid support to elute non-bound materials.Where a column is used as the solid support non-binding components ofthe GAG mixture can be eluted from the column leaving theGAG-polypeptide complexes bound to the column.

In the present invention, it is understood that certain oligosaccharidesmay interact in a non-specific manner with the polypeptide. In certainembodiments, oligosaccharide which interacts with the polypeptide in anon-specific manner may be included in, or excluded from the mixture ofcompounds enriched with one or more GAGs that modulate the effect of aheparin-binding factor. An example of a non-specific interaction is thetemporary confinement within a pocket of a suitably sized and/or shapedmolecule. Further it is understood that these oligosaccharides may elutemore slowly than those oligosaccharides that display no interaction withthe peptide at all. Furthermore it is understood that the compounds thatbind non-specifically may not require the input of the same externalstimulus to make them elute as for those compounds that bind in aspecific manner (for example through an ionic interaction). The presentinvention is capable of separating a mixture of oligosaccharides intothose components of that mixture that: bind in a specific manner to thepolypeptide; those that bind in a non-specific manner to thepolypeptide; and those that do not bind to the polypeptide. Thesedesignations are defined operationally for each GAG-peptide pair.

By varying the conditions (e.g. salt concentration) present at thesurface of the solid support where binding of the GAG and polypeptideoccurs those GAGs having the highest affinity and/or specificity for theheparin-binding domain can be selected.

GAGs may accordingly be obtained that have a high binding affinity for aprotein of interest and/or the heparin-binding domain of the protein ofinterest. The binding affinity (K_(d)) may be chosen from one of: lessthan 10 μM, less than 1 μM, less than 100 nM, less than 10 nM, less than1 nM, less than 100 pM.

GAGs obtained by the methods of the invention may be useful in a rangeof applications, in vitro and/or in vivo. The GAGs may be provided foruse in stimulation or inhibition of cell or tissue growth and/orproliferation and/or differentiation either in cell or tissue culture invitro, or in cells or tissue in vivo.

The GAGs may be provided as a formulation for such purposes. Forexample, culture media may be provided comprising a GAG obtained by themethod of the present invention.

Cells or tissues obtained from in vitro cell or tissue culture in thepresence of GAGs obtained by the method of the present invention may becollected and implanted into a human or animal patient in need oftreatment. A method of implantation of cells and/or tissues maytherefore be provided, the method comprising the steps of:

-   -   (a) culturing cells and/or tissues in vitro in contact with GAGs        obtained by the method of the present invention;    -   (b) collecting the cells and/or tissues;    -   (c) implanting the cells and/or tissues into a human or animal        subject in need of treatment.

The cells may be cultured in part (a) in contact with the GAGs for aperiod of time sufficient to allow growth, proliferation ordifferentiation of the cells or tissues. For example, the period of timemay be chosen from: at least 5 days, at least 10 days, at least 20 days,at least 30 days or at least 40 days.

In another embodiment the GAGs may be formulated for use in a method ofmedical treatment, including the prevention or treatment of injury ordisease. A pharmaceutical composition or medicament may be providedcomprising the GAGs and a pharmaceutically acceptable diluent, carrieror adjuvant. Such pharmaceutical compositions or medicaments may beprovided for the prevention or treatment of injury or disease. The useof a GAG obtained by the method of the present invention in themanufacture of a medicament for the prevention or treatment of injury ordisease is also provided. Optionally, pharmaceutical compositions andmedicaments according to the present invention may also contain theprotein of interest having the heparin-binding domain to which the GAGbinds. In further embodiments the pharmaceutical compositions andmedicaments may further comprise stem cells, e.g. mesenchymal stemcells.

Treatment of injury or disease may comprise the repair, regeneration orreplacement of cells or tissue, such as connective tissue (e.g. bone,cartilage, muscle, fat, tendon or ligament). For the repair of tissue,the pharmaceutical composition or medicament comprising the GAG may beadministered directly to the site of injury or disease in order tostimulate the growth, proliferation and/or differentiation of new tissueto effect a repair of the injury or to cure or alleviate (e.g. providerelief to the symptoms of) the disease condition. The repair orregeneration of the tissue may be improved by combining stem cells inthe pharmaceutical composition or medicament.

For the replacement of tissue, GAGs may be contacted with cells and/ortissue during in vitro culture of the cells and/or tissue in order togenerate cells and/or tissue for implantation at the site of injury ordisease in the patient. Implantation of cells or tissue can be used toeffect a repair of the injured or diseased tissue in the patient byreplacement of the injured or diseased tissue. This may involve excisionof injured/diseased tissue and implantation of new tissue prepared byculture of cells and/or tissue in contact with a GAG obtained by themethod of the present invention.

Pharmaceutical compositions and medicaments according to the presentinvention may therefore comprise one of:

-   -   (a) GAGs obtained by the method of the invention;    -   (b) GAGs obtained by the method of the invention in combination        with stem cells;    -   (c) GAGs obtained by the method of the invention in combination        with a protein containing the heparin-binding domain bound by        the GAG;    -   (d) GAGs obtained by the method of the invention in combination        with stem cells and a protein containing the heparin-binding        domain bound by the GAG;    -   (e) Tissues or cells obtained from culture of cells or tissues        in contact with GAGs obtained by the method of the invention.

GAGs isolated according to the method of the present invention may beused in the repair or regeneration of bodily tissue, especially boneregeneration, neural regeneration, skeletal tissue construction, therepair of cardio-vascular injuries and the expansion and self-renewal ofembryonic and adult stem cells. Accordingly, the GAGs may be used toprevent or treat a wide range of diseases and injuries, includingosteoarthritis, cartilage replacement, broken bones of any kind (e.g.spinal disc fusion treatments, long bone breaks, cranial defects),critical or non-union bone defect regeneration.

The use of GAGs according to the present invention in the repair,regeneration or replacement of tissue may involve use in wound healing,e.g. acceleration of wound healing, healing of scar or bone tissue andtissue grafting.

In another aspect, the present invention provides a biological scaffoldcomprising GAGs isolated by the method of the present invention. In someembodiments, the biological scaffolds of the present invention may beused in orthopaedic, vascular, prosthetic, skin and cornealapplications. The biological scaffolds provided by the present inventioninclude extended-release drug delivery devices, tissue valves, tissuevalve leaflets, drug-eluting stents, vascular grafts, wound healing orskin grafts and orthopaedic prostheses such as bone, ligament, tendon,cartilage and muscle. In a preferred embodiment of the presentinvention, the biological scaffold is a catheter wherein the inner(and/or outer) surface comprises one or more GAG compounds attached tothe catheter.

In another aspect, the present invention provides one or more GAGsisolated by the method of the present invention for use as an adjuvant.In an especially preferred aspect of the present invention, the adjuvantis an immune adjuvant.

In another aspect, the present invention provides pharmaceuticallyacceptable formulations comprising a mixture of compounds comprising oneor more GAGs, said mixture being enriched with respect to GAGs thatmodulate a heparin-binding factor.

In another aspect, the invention provides pharmaceutically acceptableformulations comprising:

-   -   (i) a mixture of compounds comprising one or more GAGs, said        mixture being enriched with respect to GAGs that modulate a        heparin-binding factor; and    -   (ii) the heparin-binding factor,        for separate, simultaneous or sequential administration. In a        preferred embodiment the formulation comprises the mixture of        compounds comprising one or more GAGs, said mixture being        enriched with respect to GAGs that modulate a heparin-binding        factor and the heparin-binding factor in intimate admixture, and        is administered simultaneously to a patient in need of        treatment. In a further embodiment of the invention, the        formulation comprises the mixture of compounds comprising one or        more GAGs, said mixture being enriched with respect to GAGs that        modulate BMP-2 and BMP-2 in intimate admixture, and is        administered simultaneously to a patient in need of treatment.

In another aspect of the present invention a kit is provided for use inthe repair, or regeneration of tissue, said kit comprising (i) apredetermined amount of a GAG having high affinity for a protein havinga heparin-binding domain, and (ii) a predetermined amount of the proteinhaving said heparin-binding domain.

In preferred embodiments the GAG is HS/BMP2 and the protein having theheparin-binding domain is BMP2.

The compounds of the enriched mixtures of the present invention can beadministered to a subject as a pharmaceutically acceptable salt thereof.For example, base salts of the compounds of the enriched mixtures of thepresent invention include, but are not limited to, those formed withpharmaceutically acceptable cations, such as sodium, potassium, lithium,calcium, magnesium, ammonium and alkylammonium. The present inventionincludes within its scope cationic salts, for example the sodium orpotassium salts.

It will be appreciated that the compounds of the enriched mixtures ofthe present invention which bear a carboxylic acid group may bedelivered in the form of an administrable prodrug, wherein the acidmoiety is esterified (to have the form —CO2R′). The term “pro-drug”specifically relates to the conversion of the —OR′ group to a —OH group,or carboxylate anion therefrom, in vivo. Accordingly, the prodrugs ofthe present invention may act to enhance drug adsorption and/or drugdelivery into cells. The in vivo conversion of the prodrug may befacilitated either by cellular enzymes such as lipases and esterases orby chemical cleavage such as in vivo ester hydrolysis.

Medicaments and pharmaceutical compositions according to aspects of thepresent invention may be formulated for administration by a number ofroutes, including but not limited to, injection at the site of diseaseor injury. The medicaments and compositions may be formulated in fluidor solid form. Fluid formulations may be formulated for administrationby injection to a selected region of the human or animal body.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to show benefit to the individual. The actualamount administered, and rate and time-course of administration, willdepend on the nature and severity of the injury or disease beingtreated. Prescription of treatment, e.g. decisions on dosage etc, iswithin the responsibility of general practitioners and other medicaldoctors, and typically takes account of the disorder to be treated, thecondition of the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams &Wilkins.

In this specification a patient to be treated may be any animal orhuman. The patient may be a non-human mammal, but is more preferably ahuman patient. The patient may be male or female.

Methods according to the present invention may be performed in vitro orin vivo, as indicated. The term “in vitro” is intended to encompassprocedures with cells in culture whereas the term “in vivo” is intendedto encompass procedures with intact multi-cellular organisms.

Stem Cells

Cells contacted with GAGs obtained by the method of the presentinvention include stem cells.

The stem cells cultured and described herein may be stem cells of anykind. They may be totipotent or multipotent (pluripotent). They may beembryonic or adult stem cells from any tissue and may be hematopoieticstem cells, neural stem cells or mesenchymal stem cells. Preferably theyare adult stem cells. More preferably they are adult mesenchymal stemcells, e.g. capable of differentiation into connective tissue and/orbone cells such as chondrocytes, osteoblasts, myocytes and adipocytes.The stem cells may be obtained from any animal or human, e.g. non-humananimals, e.g. rabbit, guinea pig, rat, mouse or other rodent (includingcells from any animal in the order Rodentia), cat, dog, pig, sheep,goat, cattle, horse, non-human primate or other non-human vertebrateorganism; and/or non-human mammalian animals; and/or human. Optionallythey are non-human.

In this specification, by stem cell is meant any cell type that has theability to divide (i.e. self-renew) and remain totipotent or multipotent(pluripotent) and give rise to specialized cells if so desired.

Stem cells cultured in the present invention may be obtained or derivedfrom existing cultures or directly from any adult, embryonic or fetaltissue, including blood, bone marrow, skin, epithelia or umbilical cord(a tissue that is normally discarded).

The multipotency of stem cells may be determined by use of suitableassays. Such assays may comprise detecting one or more markers ofpluripotency, e.g. alkaline phosphatase activity, detection of RUNX2,osterix, collagen I, II, IV, VII, X, osteopontin, Osteocalcin, BSPII,SOX9, Aggrecan, ALBP, CCAAT/enhancer binding protein-α (C/EBPα),adipocyte lipid binding protein (ALBP), alkaline phosphatise (ALP), bonesialoprotein 2, (BSPII), Collagen2a1 (CoII2a) and SOX9.

Mesenchymal stem cells or human bone marrow stromal stem cells aredefined as pluripotent (multipotent) progenitor cells with the abilityto generate cartilage, bone, muscle, tendon, ligament and fat. Theseprimitive progenitors exist postnatally and exhibit stem cellcharacteristics, namely low incidence and extensive renewal potential.These properties in combination with their developmental plasticity havegenerated tremendous interest in the potential use of mesenchymal stemcells to replace damaged tissues. In essence mesenchymal stem cellscould be cultured to expand their numbers then transplanted to theinjured site or after seeding in/on scaffolds to generate appropriatetissue constructs.

Thus, an alternative approach for skeletal, muscular, tendon andligament repair is the selection, expansion and modulation of theappropriate progenitor cells such as osteoprogenitor cells in the caseof bone in combination with a conductive or inductive scaffolds tosupport and guide regeneration together with judicious selection ofspecific tissue growth factors.

Human bone marrow mesenchymal stem cells can be isolated and detectedusing selective markers, such as STRO-I, from a CD34+ fractionindicating their potential for marrow repopulation. These cell surfacemarkers are only found on the cell surface of mesenchymal stem cells andare an indication of the cells pluripotency.

In yet a further aspect of the present invention, a pharmaceuticalcomposition comprising stem cells generated by any of the methods of thepresent invention, or fragments or products thereof, is provided. Thepharmaceutical composition useful in a method of medical treatment.Suitable pharmaceutical compositions may further comprise apharmaceutically acceptable carrier, adjuvant or diluent.

In another aspect of the present invention, stem cells generated by anyof the methods of the present invention may be used in a method ofmedical treatment, preferably, a method of medical treatment is providedcomprising administering to an individual in need of treatment atherapeutically effective amount of said medicament or pharmaceuticalcomposition.

Stem cells obtained through culture methods and techniques according tothis invention may be used to differentiate into another cell type foruse in a method of medical treatment. Thus, the differentiated cell typemay be derived from, and may be considered as a product of, a stem cellobtained by the culture methods and techniques described which hassubsequently been permitted to differentiate. Pharmaceuticalcompositions may be provided comprising such differentiated cells,optionally together with a pharmaceutically acceptable carrier, adjuvantor diluent. Such pharmaceutical composition may be useful in a method ofmedical treatment.

Glycosaminglycans

As used herein, the terms ‘glycosaminoglycan’ and ‘GAG’ are usedinterchangeably and are understood to refer to the large collection ofmolecules comprising an oligosaccharide, wherein one or more of thoseconjoined saccharides possess an amino substituent, or a derivativethereof. Examples of GAGs are chondroitin sulfate, keratin sulfate,heparin, dermatan sulfate, hyaluronate and heparan sulfate. Heparansulfates are preferred embodiments of the present invention.

As used herein, the term ‘GAG’ also extends to encompass those moleculesthat are GAG conjugates. An example of a GAG conjugate is aproteoglycosaminoglycan (PGAG, proteoglycan) wherein a peptidiccomponent is covalently bound to an oligosaccharide component.

In the present invention, it is understood that there are a large numberof sources of GAG compounds including natural, synthetic orsemi-synthetic. A preferred source of GAGs is biological tissue. Apreferred source of GAGs is a stem cell. An especially preferred sourceof GAGs is a stem cell capable of differentiating into a cell thatcorresponds to a tissue that will be the subject of treatment. Forexample, GAGs can be sourced from preosteoblasts for use in boneregeneration or skeletal tissue construction. In an especially preferredembodiment of the present invention, GAGs may be sourced from animmortalised cell line. In a further preferred embodiment of the presentinvention, GAGs may be sourced from an immortalised cell line which isgrown in a bioreactor. Another preferred source of GAGs is a syntheticsource. In this respect, GAGs may be obtained from the syntheticelaboration of commercially available starting materials into morecomplicated chemical form through techniques known, or conceivable, toone skilled in the art. An example of such a commercially availablestarting material is glucosamine. Another preferred source of GAGs is asemi-synthetic source. In this respect, synthetic elaboration of anatural starting material, which possesses much of the complexity of thedesired material, is elaborated synthetically using techniques known, orconceivable, to one skilled in the art. Examples of such a naturalstarting material are chitin and dextran, and examples of the types ofsynthetic steps that may elaborate that starting material, into a GAGmixture suitable for use in the present invention, are amide bondhydrolysis, oxidation and sulfation. Another example of a semi-syntheticroute to GAGs of the desired structure comprises the syntheticinterconversion of related GAGs to obtain GAGs suitable for use in thepresent invention.

Heparan Sulphate (HS)

In preferred aspects of the invention the glycosaminoglycan orproteoglycan is preferably a heparan sulfate.

Heparan sulfate proteoglycans (HSPGs) represent a highly diversesubgroup of proteoglycans and are composed of heparan sulfateglycosaminoglycan side chains covalently attached to a protein backbone.The core protein exists in three major forms: a secreted form known asperlecan, a form anchored in the plasma membrane known as glypican, anda transmembrane form known as syndecan. They are ubiquitous constituentsof mammalian cell surfaces and most extracellular matrices. There areother proteins such as agrin, or the amyloid precursor protein, in whichan HS chain may be attached to less commonly found cores.

“Heparan Sulphate” (“Heparan sulfate” or “HS”) is initially synthesisedin the Golgi apparatus as polysaccharides consisting of tandem repeatsof D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc). Thenascent polysaccharides may be subsequently modified in a series ofsteps: N-deacetylation/N-sulfation of GlcNAc, C5 epimerisation of GlcAto iduronic acid (IdoA), O-sulphation at C2 of IdoA and GlcA,O-sulphation at C6 of N-sulphoglucosamine (GlcNS) and occasionalO-sulphation at C3 of GlcNS. N-deacetylation/N-sulphation, 2-O-, 6-O-and 3-O-sulphation of HS are mediated by the specific action of HSN-deacetylase/N-sulfotransferase (HSNDST), HS 2-O-sulfotransferase(HS2ST), HS 6-O-sulfotransferase (HS6ST) and HS 3-O-sulfotransferase,respectively. At each of the modification steps, only a fraction of thepotential substrates are modified, resulting in considerable sequencediversity. This structural complexity of HS has made it difficult todetermine its sequence and to understand the relationship between HSstructure and function.

Heparan sulfate side chains consist of alternately arranged D-glucuronicacid or L-iduronic acid and D-glucosamine, linked via (1->4) glycosidicbonds. The glucosamine is often N-acetylated or N-sulfated and both theuronic acid and the glucosamine may be additionally O-sulfated. Thespecificity of a particular HSPG for a particular binding partner iscreated by the specific pattern of carboxyl, acetyl and sulfate groupsattached to the glucosamine and the uronic acid. In contrast to heparin,heparan sulfate contains less N- and O-sulfate groups and more N-acetylgroups. The heparan sulfate side chains are linked to a serine residueof the core protein through a tetrasaccharide linkageeglucuronosyl-β-(1→3)-galactosyl-β-(1→3)-galactosyl-β-(1→4)-xylosyl-β-1-O-(Serine))region.

Both heparan sulfate chains and core protein may undergo a series ofmodifications that may ultimately influence their biological activity.Complexity of HS has been considered to surpass that of nucleic acids(Lindahl et al, 1998, J. Biol. Chem. 273, 24979; Sugahara and Kitagawa,2000, Curr. Opin. Struct. Biol. 10, 518). Variation in HS species arisesfrom the synthesis of non-random, highly sulfated sequences of sugarresidues which are separated by unsulfated regions of disaccharidescontaining N-acetylated glucosamine. The initial conversion ofN-acetylglucosamine to N-sulfoglucosamine creates a focus for othermodifications, including epimerization of glucuronic acid to iduronicacid and a complex pattern of O-sulfations on glucosamine or iduronicacids. In addition, within the non-modified, low sulfated, N-acetylatedsequences, the hexuronate residues remain as glucuronate, whereas in thehighly sulfated N-sulfated regions, the C-5 epimer iduronatepredominates. This limits the number of potential disaccharide variantspossible in any given chain but not the abundance of each. Mostmodifications occur in the N-sulfated domains, or directly adjacent tothem, so that in the mature chain there are regions of high sulfationseparated by domains of low sulfation (Brickman et al. (1998), J. Biol.Chem. 273(8), 4350-4359, which is herein incorporated by reference inits entirety).

It is hypothesized that the highly variable heparan sulfate chains playkey roles in the modulation of the action of a large number ofextracellular ligands, including regulation and presentation of growthand adhesion factors to the cell, via a complicated combination ofautocrine, juxtacrine and paracrine feedback loops, so controllingintracellular signaling and thereby the differentiation of stem cells.For example, even though heparan sulfate glycosaminoglycans may begenetically described (Alberts et al. (1989) Garland Publishing, Inc,New York & London, pp. 804 and 805), heparan sulfate glycosaminoglycanspecies isolated from a single source may differ in biological activity.As shown in Brickman et al, 1998, Glycobiology 8, 463, two separatepools of heparan sulfate glycosaminoglycans obtained fromneuroepithelial cells could specifically activate either FGF-1 or FGF-2,depending on mitogenic status. Similarly, the capability of a heparansulfate (HS) to interact with either FGF-1 or FGF-2 is described in WO96/23003. According to this patent application, a respective HS capableof interacting with FGF-1 is obtainable from murine cells at embryonicday from about 11 to about 13, whereas a HS capable of interacting withFGF-2 is obtainable at embryonic day from about 8 to about 10.

As stated above HS structure is highly complex and variable between HS.Indeed, the variation in HS structure is considered to play an importantpart in contributing toward the different activity of each HS inpromoting cell growth and directing cell differentiation. The structuralcomplexity is considered to surpass that of nucleic acids and althoughHS structure may be characterised as a sequence of repeatingdisaccharide units having specific and unique sulfation patterns at thepresent time no standard sequencing technique equivalent to thoseavailable for nucleic acid sequencing is available for determining HSsequence structure. In the absence of simple methods for determining adefinitive HS sequence structure HS molecules are positively identifiedand structurally characterised by skilled workers in the field by anumber of analytical techniques. These include one or a combination ofdisaccharide analysis, tetrasaccharide analysis, HPLC and molecularweight determination. These analytical techniques are well known to andused by those of skill in the art.

Two techniques for production of di- and tetra-saccharides from HSinclude nitrous acid digestion and lyase digestion. A description of oneway of performing these digestion techniques is provided below, purelyby way of example, such description not limiting the scope of thepresent invention.

Nitrous Acid Digestion

Nitrous acid based depolymerisation of heparan sulphate leads to theeventual degradation of the carbohydrate chain into its individualdisaccharide components when taken to completion.

For example, nitrous acid may be prepared by chilling 250 μl of 0.5 MH₂SO_(4|) and 0.5 M Ba(NO₂)₂ separately on ice for 15 min. Aftercooling, the Ba(NO₂)₂ is combined with the H₂SO₄ and vortexed beforebeing centrifuged to remove the barium sulphate precipitate. 125 μl ofHNO₂ was added to GAG samples resuspended in 20 μl of H₂O, and vortexedbefore being incubated for 15 min at 25° C. with occasional mixing.After incubation, 1 M Na₂CO₃ was added to the sample to bring it to pH6. Next, 100 μl of 0.25 M NaBH₄ in 0.1 M NaOH is added to the sample andthe mixture heated to 50° C. for 20 min. The mixture is then cooled to25° C. and acidified glacial acetic acid added to bring the sample to pH3. The mixture is then neutralised with 10 M NaOH and the volumedecreased by freeze drying. Final samples are run on a Bio-Gel P-2column to separate di- and tetrasaccharides to verify the degree ofdegradation.

Lyase Digestion

Heparinise III cleaves sugar chains at glucuronidic linkages. The seriesof Heparinase enzymes (I, II and III) each display relatively specificactivity by depolymerising certain heparan sulphate sequences atparticular sulfation recognition sites. Heparinase I cleaves HS chainswith NS regions along the HS chain. This leads to disruption of thesulphated domains. Heparinase III depolymerises HS with the NA domains,resulting in the separation of the carbohydrate chain into individualsulphated domains. Heparinase II primarily cleaves in the NA/NS“shoulder” domains of HS chains, where varying sulfation patterns arefound. Note: The repeating disaccharide backbone of the heparan polymeris a uronic acid connected to the amino sugar glucosamine. “NS” meansthe amino sugar is carrying a sulfate on the amino group enablingsulfation of other groups at C2, C6 and C3. “NA” indicates that theamino group is not sulphated and remains acetylated.

For example, for depolymerisation in the NA regions using Heparinase IIIboth enzyme and lyophilised HS samples are prepared in a buffercontaining 20 mM Tris-HCL, 0.1 mg/ml BSA and 4 mM CaCl₂ at pH 7.5.Purely by way of example, Heparinase III may be added at 5 mU per 1 μgof HS and incubated at 37° C. for 16 h before stopping the reaction byheating to 70° C. for 5 min.

Di- and tetrasaccharides may be eluted by column chromatography.

Heparin-Binding Domains

Cardin and Weintraub (Molecular Modeling of Protein-GlycosaminoglycanInteractions, Arteriosclerosis Vol. 9 No. 1 January/February 1989 p.21-32), incorporated herein in entirety by reference, describesconsensus sequences for polypeptide heparin-binding domains. Theconsensus sequence has either a stretch of di- or tri-basic residuesseparated by two or three hydropathic residues terminated by one or morebasic residues. Two particular consensus sequences were identified:XBBXBX [SEQ ID NO: 15] and XBBBXXBX [SEQ ID NO: 16] in which B is abasic residue (e.g. Lysine, Arginine, Histidine) and X is a hydropathicresidue (e.g. Alanine, Glycine, Tyrosine, Serine). Heparin-bindingdomains are reported to be abundant in amino acids Asn, Ser, Ala, Gly,Ile, Leu and Tyr and have a low occurrence of amino acids Cys, Glu, Asp,Met, Phe and Trp.

These consensus sequences may be used to search protein or polypeptideamino acid sequences in order to identify candidate heparin-bindingdomain amino acid sequences which may be synthesised and tested for GAGbinding in accordance with the present invention.

WO 2005/014619 A2 also discloses numerous heparin-binding peptides. Thecontents of WO 2005/014619 A2 are incorporated herein in entirety byreference.

Proteins Containing Heparin-Binding Domains

The following proteins are known to contain heparin-binding domains, andpolypeptides derived from the amino acid sequences of these proteins maybe used for the identification of GAGs according to the presentinvention.

Mitogens/Morphogens/Chemokines

Fibroblast Growth Factors (FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6,FGF-7, FGF-8, FGF-9) as well as the FGF receptors FGFR1, FGFR2, FGFR3;HGF (Hepatocyte growth factor); VEGFs (Vascular endothelial growthfactor); Activins; BMPs (Bone morphogenetic protein, e.g. BMP-2, BMP-4);TGF-βs (Transforming growth factor); PDGFs (Platelet-derived growthfactor); OPG (Osteoprotegerin); HB-GAM (Heparin-bindinggrowth-associated molecules); pleiotropins; GM-CSF(Granulocyte-macrophage colony-stimulating factor); Interferon-χ; NT4/5(Neurotophin); GDNF (Glial cell-derived neurotrophic factor); WntsHedgehogs.

Antagonists

Noggin, Chordin, Sclerostin, CTGF (Connective Tissue Growth Factor),Follistatin, Gremlin.

Adhesive Glycoproteins

Fibronectin, Vitronectin, Laminin, Collagens, Thrombospondin, Tenascin,vonWillebrand Factor, NCAM (Neural Cell Adhesion Molecule), N-cadherin

Enzymes

Lipoprotein Lipase, Hepatic Lipase, Phospholipase, Apolipoprotein B,Apolipoprotein E.

Swine Protease Inhibitors

Antithrombin III, Heparin Co-factor II, Protease Nexins

Other Factors

Superoxide Dimustase, Elastase, Platelet Factor 4, N-CAM, TranscriptionFactors, DNA Topoisomerase, RNA Polymerase, Tumor Necrosis Factor.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIG. 1. Anion exchange chromatography of MX samples disrupted using 8 MUrea/CHAPS buffer. A large GAG peak is observed after 1M NaCl elution.

FIG. 2. Representative chromatogram of the desalting system duringMX-derived GAG purification. The initial peak (12-18 min) representsfull length GAG chains. The conductivity peak and debris peak (19-30min) represent salt and GAG debris elution.

FIG. 3. tGAGs (2.5 mg) loaded onto an underivatised Hi-Trap streptavidincolumn. All GAGs elute from the column in the flowthrough, indicating no“background” attachment of GAGs to the column.

FIG. 4. BMP2-HBP (1 mg) pre-incubated with tGAGs (25 mg) for 30 min.Elution profile shows the peptide (280 nm) exiting the column in theflowthrough together with the tGAG sample.

FIG. 5. BMP2-HBP (1 mg) loaded onto a Hi-Trap column. The 280 nmabsorbance levels indicate that the peptide remains attached to thecolumn even under high salt conditions; thus there was successfulcoupling of the biotinylated peptide to the streptavidin linker.

FIG. 6. BMP2-HBP (1 mg) coupled column loaded with of 25 mg of tGAGs.The chromatogram (232 nm) clearly shows both an overloading of thecolumn, in the flow through as well as the binding of some GAGs to theBMP2-HBP bed.

FIG. 7. Re-application of the GAG− (flowthrough) fractions from theprevious experiment (FIG. 6). The presence of a significant GAG+ elutionpeak indicates that all available BMP2-HBP binding sites had beensaturated, resulting in a large proportion of susceptible GAGs exitingthe column in the flowthrough.

FIG. 8. BMP2-HBP (2 mg) coupled column loaded with tGAGs (6 mg). Thechromatogram (232 nm) clearly shows no overloading of the column, andthe presence of a GAG subpopulation with a relative affinity for theBMP2-HBP.

FIG. 9. Re-run of GAG− (flowthrough) from previous run (FIG. 8). Theabsence of a GAG+ elution peak indicates that the available BMP2-HBPbinding sites were not saturated in the previous run, allowing theefficient extraction of GAG+ sugars in a single run.

FIG. 10. Re-application of isolated full length GAG+ fractions (2 mg)shows no change in affinity for the BMP2-HBP (2 mg) column prior toheparinase III digestion. A reapplication of GAG− fractions against theBMP2-HBP column also showed no change in affinity, with all GAGs exitingthe column in the flowthrough essentially as in FIG. 9.

FIG. 11. GAG− fractions (1 mg) digested with heparinase III beforeloading onto the BMP2-HBP (2 mg) column. The chromatogram (232 nm)clearly shows that no GAG samples remain bound to the column, but exitin the flowthrough. This indicates the absence of any GAG+ domains inthe full length GAG− chains.

FIG. 12. GAG+ fractions (2 mg) digested with heparinase 3 before loadingonto the BMP2-HBP (2 mg) column. The chromatogram (232 nm) demonstratesthat the GAG+ samples are retained by the column, suggesting that alldomains on the full length GAG+ chain have a relative affinity for theBMP2-HBP. The increase in the absorbance peak, as compared to the samedry weight quantity of GAG+ (FIG. 10), indicates the efficacy of theheparinase 3 treatment.

FIG. 13. Full length GAG+ chains separated using a Biogel P10 columnwith an exclusion limit of between 1.5 kDa and 20 kDa. The chromatogramshows that a large proportion of the sample chains have an overallmolecular weight of more than 20 kDa.

FIG. 14. Full length GAG+ sugar chains treated with nitrous acid for 20min to diagnostically degrade heparan sulfate species. The chromatogram,generated from a Biogel P10 sizing column, shows an almost completedegradation of all GAG+chains as compared to FIG. 13, indicating thatGAG+ isolated chains consist primarily of heparan sulfate.

FIG. 15. Chondroitin-4-sulfate (6 mg) loaded onto BMP2-HBP (2 mg)column. The chromatogram clearly illustrates a significant proportion ofthe GAG chains having an affinity for the peptide, as they eluted at asimilar salt concentration as the GAG+samples.

FIG. 16. Chondroitin-6-sulfate (6 mg) loaded onto BMP2-HBP (2 mg)column. The chromatogram indicates that few of the C6S GAG chains haveany affinity for the peptide column.

FIG. 17. Dermatan sulfate (6 mg) loaded onto the BMP2-HBP (2 mg)affinity column. The chromatogram indicates that few of the DS GAGchains had any affinity for the peptide, with only a small proportion ofthe GAGs being eluted at a similar salt concentration to GAG+ samples.

FIG. 18. Bovine heparan sulfate (2.5 mg) loaded onto the BMP2-HBP (2 mg)column. The chromatogram (232 nm) reveals only a small fraction of theGAGs binding to the column.

FIG. 19. Heparin-LMW (50 mg) loaded onto the BMP2-HBP (2 mg) column. Thechromatogram (232 nm) reveals that almost no GAG bound to the peptide.

FIG. 20. Heparin-HMW (28 mg) loaded onto the BMP2-HBP (2 mg) column. Thechromatogram (232 nm) reveals that almost no GAG bound to the peptide.

FIG. 21. Heparin-HMW (25 mg) predigested with heparinase I was loadedonto the BMP2-HBP (2 mg) column. The chromatogram (232 nm) reveals thatvery few GAG fragments bound to the peptide.

FIG. 22. Chromatogram showing steps in isolation of BMP-2 peptidespecific HS by affinity chromatography.

FIG. 23. Chromatogram showing elution of BMP-2 peptide specific HS(GAG+) by affinity chromatography.

FIG. 24. Chromatogram showing elution of BMP-2 peptide non-specific HS(GAG−) by affinity chromatography.

FIG. 25. Chromatogram showing elution of Sigma HS(H9902) standard undersize exclusion chromatography on Superdex 75 column.

FIG. 26. Chromatogram showing elution of BMP-2 peptide specific HS(GAG+) under size exclusion chromatography on Superdex 75 column.

FIG. 27. Graph showing Osterix expression in C2C12 cells in response tocontrol media, 100 ng/ml BMP2 and 300 ng/ml BMP2.

FIG. 28. Graph showing Osteocalcin expression in C2C12 cells in responseto control media, 100 ng/ml BMP2 and 300 ng/ml BMP2.

FIG. 29. Graph showing Runx2 expression in C2C12 cells in response tocontrol media, 100 ng/ml BMP2 and 300 ng/ml BMP2.

FIG. 30. Graph showing expression of Alkaline Phosphatase as measured byquantitative PCR in C2C12 cells in response to control media, BMP-2,Negative GAG (GAG−), Positive GAGs (GAG+), Total HS and Heparin (Hep).

FIG. 31. Graph showing expression of Osterix as measured by quantitativePCR in C2C12 cells in response to control media, BMP-2, Negative GAG(GAG−)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total HS and Heparin (Hep).

FIG. 32. Graph showing expression of BspII as measured by quantitativePCR in C2C12 cells in response to control media, BMP-2, Negative GAG(GAG−)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total HS and Heparin (Hep).

FIG. 33. Graph showing expression of Runx2 as measured by quantitativePCR in C2C12 cells in response to control media, BMP-2, Negative GAG(GAG−)+BMP-2, Positive GAGs (GAG+)+BMP-2, Total HS and Heparin (Hep).

FIG. 34. Graph showing expression of Osteocalcin in C2C12 cells inresponse to BMP and GAG+ (+BMP-2) isolated from MC3T3-E1 cells.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below including specific details of thebest mode contemplated by the inventors for carrying out the invention,by way of example. It will be apparent to one skilled in the art thatthe present invention may be practiced without limitation to thesespecific details.

We investigated the potential of GAGs to augment the activities of bonemorphogenic protein 2 (BMP2). The highly osteoinductive activity of BMP2for the murine myogenic cell line C2C12 have been well characterised.Studies both in this cell line, and in vivo, have implicated a role forglycosaminoglycans in modulating this activity.

BMP2's affinity for heparin has similarly been well characterised.Numerous studies have been conducted that have sought to examine thedynamic interaction between BMP2 and GAGs. Some have proposed that theinteraction is inhibitory, and so responsible for either sequesteringthe cytokine away from the receptor or inducing its association with itsnumerous inhibitors, such as noggin, that have been shown, similarly, tohave an affinity for heparin. Alternative findings implicate theinteraction between BMP2 and GAGs is one of maintaining a localconcentration of the cytokine around cells that require its signallingin order to differentiate into the osteoblast lineage.

These findings also suggest that the association serves to significantlylengthen the half-life of the homodimer, so allowing it to remain activein the ECM for longer periods. As is the case with most systems, theactual role of this interaction is likely to be blend of some, or all ofthe above.

Although many studies have provided evidence for the interaction thatBMP2 has with model sugars, the specific interaction between the BMP2heparin-binding peptide (BMP2-HBP), a string of amino acids(QAKHKQRKRLKSSCKRHP [SEQ ID NO: 1]) located at the N-terminal end ofeach BMP2 monomer, and appropriate glycosaminoglycans has receivedrelatively little attention. A major question that arises is whetherthere is a complementary saccharide sequence embedded within an HS chainthat controls the association with an absolute, or at least relative,specificity.

We sought to isolate a sequence-specific glycosaminoglycan that couldmodulate BMP2 activity via a direct interaction with the cytokine.

Example 1 Materials and Methods Buffer Preparation

Preparation of all buffers for GAG extraction and analysis is conductedwith strict attention paid to quality. It is vital that the pH ofbuffers is maintained at the correct level and that all buffers befiltered and degassed in order to prevent the clogging of columns withprecipitates or bubbles. The formation of bubbles, in particular, cancause serious damage to columns, and in the case of sealed,pre-fabricated columns, leads to them becoming unusable.

All buffers used were filtered with 1×PBS without Ca²⁺ or Mg²⁺ (150 mMNaCl), or double distilled (ddH₂O) to make the final solutions.

Disruption Buffer

The 8M Urea/CHAPS disruption buffer consisted of PBS (150 mM NaCl) with1% CHAPS, 8M Urea and 0.02% NaN₃ to prevent contamination by microbialgrowth during storage. This solution was used to disrupt matrix (MX)samples, so was not degassed or filtered.

PGAG Anion Exchange Low Salt (250 mM) Buffer

Low salt PGAG anion exchange buffer comprised PBS (150 mM NaCl) with anadditional 100 mM NaCl. The buffer was equilibrated to pH 7.3 with NaOHand 0.02% NaN₃. The solution was then degassed under negative pressureand constant stirring until no further bubbles were released beforebeing filtered through a 0.4 μm filter.

PGAG Anion Exchange High Salt (1M) Buffer

High salt PGAG anion exchange buffer comprised PBS (150 mM NaCl) with anadditional 850 mM NaCl. The buffer was equilibrated to pH 7.3 with NaOHand 0.02% NaN₃ added. The solution was then degassed under negativepressure and constant stirring before being filtered through a 0.4 μmfilter.

Pronase/Neuraminidase PGAG Reconstitution Buffer

This buffer was used to reconstitute desalted PGAG samples after anionexchange in order to prepare them for enzymatic digestion of theassociated core proteins. It consisted of 25 mM sodium acetate(CH₃COOHNa). The buffer was equilibrated to pH 5.0 with glacial aceticacid (CH₃COOH). Both pronase and neuraminidase enzymes werereconstituted according to the manufacturer's instructions.

GAG Affinity Chromatography Low Salt (150 mM) Buffer

Low salt GAG anion exchange buffer was made using PBS (150 mM NaCl)without any additional salt. The buffer was equilibrated to pH 7.3 withNaOH and 0.02% NaN₃. The solution was degassed under negative pressureand constant stirring until no further bubbles were released beforebeing filtered through a 0.4 μm filter.

GAG Affinity Chromatography High Salt (1M) Buffer

High salt GAG anion exchange buffer was made using PBS (150 mM NaCl)with an additional 850 mM NaCl. The buffer was equilibrated to pH 7.3with NaOH and 0.02% NaN₃ was added, the solution was then degassed andfiltered through a 0.4 μm filter.

Desalting Solution

The desalting solution was made using ddH₂O that was equilibrated to pH7.0 with 0.02% NaN₃. The solution was then degassed and filtered.

Sample Preparation

Matrix samples were disrupted using Disruption Buffer (8M Urea/CHAPS),then scraped off the culture surface in this buffer and stirredovernight at 37° C. to ensure maximal lysis. The samples were thencentrifuged at 5000 g for 30 min and the supernatant was clarifiedthrough a 0.4 μm filter in preparation for PGAG extraction via anionexchange chromatography.

Column Preparation & Usage

The choice and preparation of the types of columns to be used for eachsequential step in the isolation and characterisation of GAGs is ofmajor importance for the success of the protocol. It was vital that ateach step the columns were equilibrated and cleaned with great care.

Anion Exchange Columns

Due to the relatively large quantities of MX substrate used for GAGextraction, and the high load this places on the column system, it wasnecessary to pack and prepare a large anion exchange column manually,specifically for this study. Capto Q anion exchange beads (Pharmacia)were packed into a Pharmacia XK 26 column (Pharmacia) to produce acolumn with a maximum loading capacity of 500 ml of MX lysate per run.

Prior to use, both the column and all buffers were equilibrated to roomtemperature for 30 min, before washing and equilibrating the column inPGAG Anion Exchange Low Salt (250 mM) Buffer for 30 min until allabsorbance channels remained stable. The clarified cell lysate was thenpassed through the column which was again rinsed in 500 ml of low saltbuffer to remove any nonspecifically bound debris. PGAGs were theneluted using 250 ml of PGAG Anion Exchange High Salt (1M) Buffer andlyophilised prior to desalting. The column was then rinsed in low saltbuffer and returned to 4° C. for storage.

Desalting Protocol

After PGAG/GAG isolation it was necessary to remove the high amount ofsalt that accumulated in the sample during elution from the column. Forthis step, all eluted samples of the same experimental group werecombined and loaded onto 4× Pharmacia HiPrep™ 26/10 desalting columns.Prior to use, both the columns and all solutions were equilibrated toroom temperature for 30 min before washing and equilibrating the columnin Desalting Solution for 30 min until all absorbance channels achievedstability. Lyophilised samples were reconstituted in Desalting Solutionin the minimum possible volume that resulted in a clear solution. Thiscombination of columns permitted the loading of up to 60 ml of sample.Those fractions eluting from the column first were lyophilised andretained for further separation or cell culture application. The columnswere then rinsed in Desalting Solution and returned to 4° C. forstorage.

BMP2-HBP Column Preparation

The isolation of GAGs carrying relative affinities for BMP2 wasconducted using a BMP2-HBP column. Approximately 2 mg of biotinylatedBMP2-HBP was prepared in 1 ml of the GAG Affinity Chromatography LowSalt (150 mM) Buffer. This amount was loaded onto a HiTrap StreptavidinHP column (Pharmacia) and allowed to attach to the column for 5 min. Thecolumn was then subjected to a complete run cycle in the absence ofGAGs. The column was washed in 13 ml of low salt buffer at a flow rateof 0.5 ml/min before being subjected to 10 ml of GAG High salt buffer at1 ml/min. Finally the column was rinsed with 10 ml of low salt buffer.During this process data was carefully monitored to ensure that nopeptide elution or column degradation was observed.

GAG+ Sample Isolation

Once the BMP2-HBP column had been prepared and tested for stabilityunder normal running conditions, it was ready to be used for theseparation of GAG+chains from tGAG (total GAG) samples. tGAG samples (6mg) were prepared in 3 ml of GAG affinity low salt (150 mM) buffer andinjected into a static loop for loading onto the column. Prior to useboth the BMP2-HBP column and all buffers were equilibrated to roomtemperature for 30 min before washing and equilibrating the column inlow salt buffer for 30 min until all absorbance channels were stable.The sample was then loaded onto the column at 0.5 ml/min and the columnand the sample rinsed in 10 ml of low salt buffer at 0.5 ml/min.Retained GAG+ samples were subsequently recovered by elution with 10 mlof high salt (1 M)

buffer and lyophilised for desalting. The column was then rinsed in 10ml of low salt buffer and stored at 4° C.

Pronase/Neuraminidase Treatment

In order to isolate GAG chains from their core proteins, they weredigested using pronase and neuraminidase. Lyophilized PGAG samples wereresuspended in a minimum volume of 25 mM sodium acetate (pH 5.0) andclarified by filtration through a 0.4 μm syringe filter. Total samplevolume was dispensed into 10 ml glass tubes in 500 μl aliquots. 500 μlof 1 mg/ml neuraminidase was added and incubated for 4 h at 37° C. Afterincubation 5 ml of 100 mM Tris-acetate (pH 8.0) was added to eachsample. An additional 1.2 ml of 10 mg/ml pronase, reconstituted in 500mM Tris-acetate, 50 mM calcium acetate (pH 8.0), was added to eachsample and incubated for 24 h at 36° C. After treatment all volumes werecombined and prepared for anion exchange processing by centrifugationand filtration.

GAG Digestion Protocols

The analysis of GAGs, including their sulfated domain sizes and relativesulfation levels, was carried out by using established protocolsincluding degradation by either nitrous acid or lyases.

Nitrous Acid Digestion

Nitrous acid-based depolymerisation of heparan sulfate leads to theeventual degradation of the carbohydrate chain into its individualdisaccharide components when taken to completion. Nitrous acid wasprepared by chilling 250 μl of 0.5 M H₂SO₄ and 0.5 M Ba(NO₂)₂ separatelyon ice for 15 min. After cooling, the Ba(NO₂)₂ was combined with theH₂SO₄ and vortexed before being centrifuged to remove the barium sulfateprecipitate. 125 μl of HNO₂ was added to GAG samples resuspended in 20μl of H₂O, and vortexed before being incubated for 15 min at 25° C. withoccasional mixing. After incubation, 1 M Na₂CO₃ was added to the sampleto bring it to pH 6. Next, 100 μl of 0.25 M NaBH₄ in 0.1 M NaOH wasadded to the sample and the mixture was heated to 50° C. for 20 min. Themixture was then cooled to 25° C. and acidified with glacial acetic acidto pH 3 in the fume hood. The mixture was then neutralised with 10 MNaOH and the volume was then decreased by freeze drying. The finalsamples were run on a Bio-Gel P-2 column to separate di- andtetrasaccharides to verify degradation.

Heparinase III Digestion

Heparinase III is an enzyme that cleaves sugar chains at glucuronidiclinkages. The series of heparinase enzymes (I, II and III) each displayrelatively specific activity by depolymerising certain heparan sulfatesequences at particular sulfation recognition sites. Heparinase Icleaves HS chains within NS regions along the chain. This leads to thedisruption of the sulfated domains that are thought to carry most of thebiological activity of HS. Heparinase III depolymerises HS within the NAdomains, resulting in the separation of the carbohydrate chain intoindividual sulfated domains. Lastly, Heparinase II primarily cleaves inthe NA/NS “shoulder” domains of HS chains, where varying sulfationpatterns are found.

In order to isolate potential active domains we focused on thedepolymerisation of GAG+ NA regions. Both the enzyme and lyophilised HSsamples were prepared in a buffer containing 20 mM Tris-HCl, 0.1 mg/mlBSA and 4 mM CaCl₂ at pH 7.5. The concentration of heparinase III addedto each sample is governed by the relative quantity of HS components inthe sample. Our analysis, via nitrous acid depolymerisation, indicatedthat the GAG+ samples consisted of predominantly HS; thus the enzyme wasused at 5 mU per 1 μg of HS. The sample was incubated at 37° C. for 16 hbefore the reaction was stopped by heating to 70° C. for 5 min. Thesample was then applied to the appropriate column system for furtheranalysis.

Cell Culture GAG Production

In order to isolate GAG species representative of developingosteoblasts, MC3T3 cells were grown in osteogenic conditions for 8 days.The cellular component was removed via incubation in a dilute solutionof 0.02 M ammonium hydroxide (NH₄OH) at 25° C. for 5 min. After 5 min,NH₄OH was removed by inversion of the culture surfaces. Treated cultureswere allowed to dry in a laminar flow cabinet overnight. The followingday the treated cultures were washed three times with sterile PBS andallowed to dry in the laminar flow cabinet. Prepared matrix cultureswere then stored under sterile conditions in 4° C. until primaryproteoglycans were liberated via treatment with disruption buffer andanion exchange chromatography.

BMP2-Specific GAG Bioactivity

C2C12 myoblasts were subcultured every 48 h, to a maximum of 15passages, by plating at 1.3×10⁴ cells/cm² in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% FCS. Osteogenic differentiation wasinduced at 2×10⁴ cells/cm² in DMEM supplemented with 5% FCS, nominatedconcentrations of recombinant human bone morphogenic protein-2 (rhBMP2)and glycosaminoglycan fractions with a positive or negative affinity forrhBMP2 (GAG+ and GAG− respectively). rhBMP2 and GAG fractions werepre-incubated for 30 min at 25° C. prior to addition to theircorresponding C2C12 cultures. The cultures were permitted to grow underthese conditions for 5 days, with media for each condition being changedevery 48 h, before mRNA samples were extracted and prepared for RQ-PCRanalysis. Real time PCR for osteocalcin expression was conduced usingthe ABI Prism 7000® sequence detection system (PerkinElmer LifeSciences). Primers and probes were designed using Primer Expresssoftware (v2.1, PE Applied Biosystems). The target probe was redesignedto incorporate LNA bases and labelled with BHQ-1 (Sigma-Proligo). Theribosomal subunit gene 18S (VIC/TAMRA) was used as an endogenouscontrol, with each condition consisting of three repeats, each tested intriplicate. The raw PCR data was analysed using the ABI SequenceDetector software. Target gene expression values were normalised to 18Sexpression prior to the calculation of relative expression units (REUs).

Results Anion Exchange Chromatography

In order to successfully extract GAGs from MX samples, it is necessaryto remove other matrix proteins that may contaminate the sample. As GAGsconstitute the most negatively charged molecules in the ECM, this ismost effectively accomplished with anion exchange chromatography.

Samples were disrupted using 8M Urea/CHAPS buffer and loaded onto theanion exchange column. Unwanted protein and ECM debris were washed fromthe column and the negatively charged GAGs eluted with 1 M NaCl. Atypical chromatogram (FIG. 1) clearly shows the flowthrough of a largeamount of nonadherent debris, as well as the clean and tight elution ofa large quantity of GAGs from the MX preparation. Thus not only doesthis result demonstrate the purification of GAGs by this method, it alsoconfirms the retention of a large number of GAGs in the ECM aftertreatment with NH₄OH.

Desalting

Virtually all chromatography methods employed to purify and analyse GAGsat various stages of processing require elution with high-salt buffers.As high salt conditions interfere with affinity-based chromatography, itis necessary to desalt samples after each stage of processing. Thisprocess is generally completed with size exclusion chromatography. Underthese conditions larger molecules, such as GAGs, exit the column beforesmall molecules, including the salt and small GAG debris. The separationof GAGs from the contaminating salt can be followed on the resultingchromatogram (FIG. 2) which also serves to confirm that the GAG chainsremained intact during the treatment process.

BMP2-HBP Column System Column Preparation

Due to the prohibitive costs involved in creating a BMP2 growth factorcolumn with commercially available reagents, we instead utilised abiotinylated preparation of the known heparin-binding domain of BMP2(BMP2-HBP). This peptide was immobilised on a Hi-Trap Streptavidin HPcolumn (1 ml) in order to specifically retain GAG chains with anaffinity for the specific heparin-binding domain peptide.

First we examined any background affinity the GAGs may have had for thenaked streptavidin column by running the total GAG (tGAG) fractionagainst a column bed devoid of BMP2-HBP (FIG. 3). Our results confirmedthat our MX derived tGAG samples carried no inherent affinity for thestreptavidin column. We further investigated two separate methods ofexposing tGAGs to the BMP2-HBP for the purpose of separating chains witha specific affinity. The peptide was either pre-incubated for 30 minwith 25 mg of tGAGs prior to loading onto the streptavidin column, orwas loaded first, with the tGAGs being run through the column bedthereafter.

Pre-incubation of tGAGs with the BMP2-HBP revealed the completeinability of the peptide to associate with the column (FIG. 4), letalone mediate any isolation of specific GAGs. When the peptide wasloaded onto the column alone, however, its association with the columnwas absolute, with effectively no elution of peptide, even under 1 Msalt conditions (FIG. 5). This high affinity association indicates thatthe biotin-streptavidin association is functioning correctly, andsuggests a possible inhibition of binding to the column, when loadedtogether with tGAGs, due to steric hindrance.

Column Loading Capacities

As the proportion of tGAGs that were likely to have a relative affinityfor the BMP2-HBP was unknown, we first sought to standardise thequantities of tGAGs loaded onto the peptide column at each run forseparation. Hi-Trap columns were prepared by immobilising 1 mg of theBMP2-HBP for the extraction of tGAGs with a specific affinity for theBMP2 heparin-binding site. This amount was selected so as to maximisethe quantity of available peptide for future experiments should columnstability become compromised over time. Instability is a significantproblem with peptide columns, with corresponding impacts on consistency.Initial attempts at loading of 25 mg of tGAGs onto a 1 mg BMP2-HBPcoupled column resulted in a clear overloading, as observed viaabsorbance at 232 nm in the flowthrough (FIG. 6). Although a significantelution peak was observed, tGAGs with affinity for the HBP were lost inthe flowthrough due to overloading. This was examined by re-running theflowthrough through the peptide column (FIG. 7). This resulted in asignificant GAG+ (elution) peak, indicating that the previous run hadsaturated the column.

Further optimisation led us to routinely load no more than 6 mg of tGAGsonto a 2 mg BMP2-HBP column. This, as evidenced by the flowthrough peak(FIG. 8) and the absence of a positive-binding fraction (FIG. 9),forestalled column overloading. The extraction of those tGAGs with anaffinity for the BMP-HBP from each sample set in a single pass allowedus, in turn, to separate GAG+ and GAG− fractions more efficiently.

GAG Domain Analysis GAG+Chain Specificity

With the establishment of a standardised protocol, we were able toreproducibly isolate GAG+ fractions for further analysis.

Given the domain structure of heparan sulfate that mediates the bindingspecificity for proteins, it is likely that multi-domain GAG chains thatbind to the column are in fact composed of a large proportion of chainwith little or no specific affinity for BMP2. Similarly, it is possiblethat chains that appeared GAG− may in fact contain domains that carrysome affinity for the BMP2-HBP. In order to examine these possibilities,it was necessary to break down the GAG chains into their componentdomains for more extensive examination.

The enzyme heparinase III (heparitinase I) cleaves HS chains primarilyin those areas flanking highly sulfated regions, thereby liberating thehighly charged, protein-associating domains that bind susceptible growthfactors, in this case the BMP2-HBP. Both GAG+ and GAG− fractions wereexposed to heparinase digestion, although neither fraction showed anychange in their affinity for the BMP2-HBP (FIG. 10).

Heparinase III digestion of both full length GAG+ and GAG− fractions wassubsequently conducted, and both digested sample sets subsequentlyloaded onto the BMP2-HBP column to assess retention affinity.

The efficacy of the heparinase digestion was validated by the increasein relative absorbance of samples of equal dry weight after enzymaticdigestion, as shown in FIGS. 10 and 12. As the monitoring of GAG chainsat 232 nm is via the sugar chain itself and, in particular, unsaturatedbonds, any cleavage along the chain's length by heparinase III,resulting in unsaturated bonds of HS fragments, leads to an increase inabsorbance.

Interestingly, heparinase digestion of full length GAG− chains yieldedno fractions carrying any notable affinity for the BMP2-HBP (FIG. 11).However, the digestion of full-length GAG+ samples similarly resulted inno fractions that lacked affinity for the BMP2-HBP (FIG. 12). Thisresult suggests that entire chains of BMP-binding GAG are producedcontaining domain repeats that have a specific affinity for the HBP.Alternatively, the HBP may not be able to yield sufficientdiscrimination between GAG+ domains with varying affinity under theseminimalist conditions.

GAG+ Composition Full Length GAG+ Sizing

In order to examine the composition of GAG+ fractions from the BMP2-HBPcolumn, we first examined their average size. This was to ensure that wewere actually separating GAG chains of reasonable length, rather thansmall fragments not carrying any specific affinity. Although any sizingof GAG chains is problematical, owing to their relatively rigid rod-likeconformation, a set of assumptions invoking Stoke's radius and apparentsphericity can be made.

Full length GAG+ samples were loaded onto Biogel P10 gel filtrationcolumns (1 cm×120 cm) with an exclusion limit of between 20 kDa to 1.5kDa. Absorbance measured at 232 nm indicated a large proportion of GAG+molecules had an overall apparent size greater than 20 kDa (FIG. 13).

It has been posited that sugar chains must be longer than approximately10-14 rings in order to potentiate significant biological activity forthe FGF family of mitogens. In terms of apparent molecular weight, achain of 14 fully sulfated disaccharides corresponds to approximately8.7 kDa. As the majority of chains found in the GAG+ samples show anapparent molecular weight >20 kDa, it is reasonable to assume that theinteraction that they carry for the BMP2-HBP has some specific affinityand is not the result of a general non-specific interaction.

GAG+ Sugar Species

There are five major glycosaminoglycan sugar families: hyaluronan,keratan sulfate, dermatan sulfate, chondroitin sulfate and heparansulfate. Of these five, only heparan sulfate, chondroitin sulfate anddermatan sulfate have the capacity to generate variably sulfated domainsthat may code for specific interactions with particular cytokines suchas BMP2. The identification of the type of sugar species isolated usingthe BMP2-HBP column was of crucial importance for this study, and wasdetermined using a combination of diagnostic chemical and enzymaticdegradations. In particular, heparan sulfate, one of the major GAGcandidates for the interaction with BMP2, can be completely degradedinto its disaccharide components in the presence of nitrous acid.

Thus, our HBP-retained GAG samples were incubated with nitrous acid for20 min prior to separation on a Biogel P10 sizing column. Examination ofthe resulting chromatogram revealed an almost complete degradation ofall GAG+ sugar samples, as measured by absorbance at 232 nm and 226 nm(FIG. 14). This result strongly suggests that the full length sugarchains isolated specifically against the BMP2-HBP consist primarily ofheparan sulfate, as other sugar chains are not affected by nitrous aciddepolymerisation.

Although almost all the GAG+ chains could be degraded in such a manner,a small peak was nevertheless observed at higher molecular weights (>20kDa). It can be postulated to consist of chondroitin sulfates, of whichCS-B (dermatan sulfate) and CS-E (chondroitin-4,6-sulfate) demonstratesulfation complexity akin to heparan sulfates.

GAG Species Analysis BMP2-HBP Specific GAGs (Alternative Species)

The degradation of full length GAG+ chains by exposure to nitrous acidclearly indicated that the majority of GAG+ sugar chains consisted ofthe heparan sulfate sugar species (FIG. 14). The degradation of the GAG+sample was not, however, complete as was observed by the remnant peak inthe high molecular weight region. The presence of this peak pointsstrongly to the possibility of other species of sugar chains, such aschrondroitin or dermatan sulfate. We next sought to examine the possibleaffinity the other two sugar types may have for this cytokine by firstexamining a variety of commercially available chondroitin and dermatansugars for their affinity to the BMP2-HBP column.

We tested chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S) anddermatan sulfate (DS) by, in each instance, loading 6 mg of the sugaronto the BMP2-HBP column under the same conditions used to isolate GAG+chains from MC3T3 matrix samples.

The chromatograms illustrating the affinity of each of the 3 sugar chaintypes showed that only C4S (FIG. 15) had any significant affinity forthe peptide. This affinity taken together with the lack of affinity forthe BMP2-HBP column observed for both C6S (FIG. 16) and DS (FIG. 17)samples, appears to indicate that C4S has a particular, potentiallysignificant, interaction with the BMP2 heparin-binding site.

As any potential interaction between chondroitin sulfate and BMP2 hasnot yet been well characterised, these results led us to question thevalidity of column chromatography as an accurate monitor of theBMP2/heparan interaction. In order to further explore the specificity ofthe interaction dynamic, we tested several commercially available sugarspecies for their affinity to the column. These included heparansulfate, low molecular weight heparin (Heparin-LMW), high molecularweight heparin (Heparin-HMW) and Heparin-HMW treated with heparinase I.

Interestingly, none of these commercially available GAG species appearedto demonstrate any specific interaction with the peptide column. Heparansulfate from bovine kidney had very little affinity (FIG. 18), abehaviour that was further confirmed by its inability to positivelyaugment FGF2-mediated cell proliferation (data not shown), as isobserved in the presence of HS2. This reduced ability of this GAG sampleto bind the column may be as a result of it being sold in a relativelyunsulfated form.

None of the tested heparin samples showed even a minor affinity for thecolumn. This is of particular interest as BMP2 itself was historicallyfirst isolated using heparin columns. In order to confirm this result,both LMW (FIG. 19) and HMW (FIG. 20) heparin were tested; neither showedany appreciable affinity for the column.

As we surmised that the relatively small BMP2-HBP peptide may have haddifficulty maintaining its association with the much larger heparinmolecules, we next predigested the heparin-HMW samples using heparinaseI. These smaller heparin-HMW fragments were then run over the BMP2-HBPcolumn; this treatment did not, however, appear to improve the abilityof any of the heparin samples to bind the peptide column (FIG. 21).

This inability of the peptide column to show any specific interactionwith any of the various preparations of heparin was somewhat unexpected,due to BMP2 conventionally being isolated via heparin affinity. It ispossible, however, that this may be as a result of the reversing of the“receptor-ligand” order of interaction; in this case the BMP2-HBPrepresented the fixed “receptor” as opposed to the heparin thatrepresented the “ligand”, or that the concentrations of BMP2-HBP orsoluble heparin favour a dissociated state that rapidly negates anyaffinity under flow/salt stress.

CONCLUSIONS

The use of a preosteoblast-derived ECM substrate provided us with auseful model for simulating the activity of natively secreted,ECM-associated GAGs in relation to such osteoinduction. Though numerousprevious studies have examined the role that this native interaction hasin modulating the activity of BMP2, this has usually been conducted atthe level of the cytokine, rather than with a view to exploring thesequence specificity of the biomodulating GAGs.

Hence here we sought to exploit the availability of natively secretedGAGs in the MX substrate and their potential for direct,sequence-specific interaction and modulation of BMP2-induced C2C12myoblast commitment to the osteogenic lineage.

Anion Exchange

The use of this particular standard and well characterised protocolprovided us with conclusive evidence for GAG accessibility from theNH₄OH-treated MX substrate. Our initial concerns were centred around theharsh chemical treatment used to lyse the cellular components of theECM, and that this may have also resulted in the stripping of themajority of GAGs from the ECM. However, the significant, high affinitypeak observed in the anion exchange chromatogram clearly illustrates theretention of a large quantity of GAGs within the MX substrate. Whilethis particular methodology does not allow for the identification ofindividual GAG species, it does offer conclusive evidence of theirpresence in the sample due to their being amongst the mostnegatively-charged molecules secreted by cells.

BMP2-HBP Column System

Previous research into the functional role of the BMP2 heparin-bindingpeptide provided us with a useful tool to investigate the potentiallyspecific interaction that BMP2 has with GAGs. This single string ofamino acids, located at the N-terminus of each BMP2 monomer, appears tobe solely responsible for mediating BMP2's affinity for GAGs.

We thus investigated the use of this region of the BMP2 molecule as aligand “bait” in attempts to retain those GAG chains that carriedrelative affinity for the cytokine. The use of the BMP2-HBP in thismanner resulted in a significant retention of HS to the peptide column(GAG+).

Column Preparation

Using an N-terminal biotinylated HBP we prepared a BMP2-HBP affinitychromatography column, and were able to successfully retain GAG samplesthat were candidates for controlling the native BMP2 homodimer. Initialpreparations of the column highlighted some interesting problems.Preparations of biotinylated BMP2-HBP that were premixed with tGAGsshowed an inability to bind to the column. As later tests showed thatthe BMP2-HBP easily attached to the streptavidin column when loaded onits own this result indicated that the GAGs interfered with the abilityof the peptide's biotinylation site to associate with the streptavidincolumn. The tGAGs themselves carried no affinity for the streptavidin,indicating that the direct interaction with the BMP2-HBP, possibly viasteric hindrance, was responsible for this.

Column Optimisation

Without any direct information that would allow us to estimate thebinding capacities of GAG+ sugars in our samples, our peptide columnneeded to be optimised to ensure that excessive sample loading would notlead to column saturation and consequent sample loss. This initiallyinvolved intentionally saturating the column in order to examine thebinding capacity of a known quantity of BMP2-HBP. Even with a largequantity of tGAGs the peptide was capable of retaining the majority ofGAG+sugar chains. Under these conditions as little as 1 mg of BMP2-HBPwas able to completely retain all GAG+ chains within two cycles. Thecolumn thus appeared to “simulate” a true BMP2 growth factor column andprovide an extremely efficient way of extracting GAG+ samples.

The optimisation of peptide-based columns for specific GAG isolation isa complex procedure that varies greatly depending on the size andindividual chemical characteristics of the protein used. Previousstudies, utilising FGF-1 and 2 growth factor columns (Turnbull andNurcombe, personal communication), also showed a significant need forcontinual column maintenance and short viable column life-spans. Thesestudies demonstrate the laborious nature of working with peptide columnsand the care that must be taken to correctly optimise this manner ofsystem. Unfortunately, while other systems for the analysis of specificprotein-GAG interactions exist, these generally lack the capacity toisolate sufficient quantities of GAGs for further analysis, making theminappropriate for our intended course of study.

GAG Domain Analysis

GAG sulfation patterns are, particularly in the case of heparan sulfate(HS), frequently concentrated into domains of high sulfation that areinterspaced with regions of little sulfation. This grouping of sulfationsites into domains is what provides region-specific binding of ligandsto the GAG chain, allowing a single sugar molecule to potentially bind avariety of different targets, and to stabilise the interaction betweenthese, as is seen in the FGF system. Exceptions to this proposed modelfor HS-ligand interactions include the interaction between interferongamma (IFNγ) and heparan sulfate. In this instance the interactionbetween the GAG and IFNγ leads to an increased potency of the cytokine.IFNγ that remains dissociated from local GAGs is rapidly processed intoan inactive form, thereby preventing its signalling in inappropriateareas after diffusion. IFNγ also displays four separate heparin-bindingdomains, each with a different sequence, a finding not unusual forheparin-binding proteins. However, only two domains found immediately atthe C-terminus of the protein have been shown to mediate INFγ'sheparin-binding characteristics. Importantly, sequence analysis of theHS sequence with specific affinity for these two IFNγ heparin-bindingsites revealed an interesting difference in comparison to the commonlyobserved model of HS-ligand interaction. In this case, the sequence ofHS responsible for the binding of IFNγ was found to be composed of apredominantly N-acetylated region, carrying little sulfation. Thisregion was flanked by two small N-sulfated regions. This differssignificantly with the system observed in FGF, where sulfation patternsin NS domains are responsible for mediating the interaction between FGFand HS. In recent years, this type of interaction has been observed innumerous other systems, such as PDGF, IL-8 and endostatin. The discoveryof this kind of interaction with HS, as observed in these cytokines, maybe able to explain the bioactivity observed in hyaluronan, which carriesno sulfation patterns at any point along its chain and yet has theability to modulate the activity of such factors as NF-κB.

These observed interactions between ligands and GAGs, in particular thatof IFNγ, differ significantly to the proposed, and our observed, mode ofinteraction between HS and BMP2. BMP2's single, N-terminalheparin-binding domain exhibits no secondary structure and appears tointeract with HS solely on the basis of charge. While in-depth sequenceanalysis of HS that binds this peptide sequence was not conducted, itsrequirement to be eluted under approximately 300 mM NaCl conditions leadus to suspect the presence of a moderate degree of sulfation, therebyplacing this interaction within the conventional model of sulfationpatterns mediating specific interactions.

GAG+ Chain Specificity

The allocation of sulfation patterns into domains that give HS itsability to stabilise proteomic interactions also results in thepossibility that a GAG+ sugar chain of sufficient length and complexitymay carry several domains that have no direct affinity for the BMP2-HBPon their own, due to their carrying a different sulfation sequence.Conversely, it is also possible that some full-length sugar chains thatwere identified as having little affinity for the BMP2-HBP (GAG−) maycontain some cryptic domains that do carry such affinity.

In recent years, numerous reports have been published that providestrong evidence for a “sulfation code” within these complex carbohydratechains. While the details of this “sulfation code” remain difficult toelucidate, and the sequencing of long chains of sulfated carbohydratesis a complex and time consuming process, a number of possible modes ofspecific interaction between GAGs and ligands have been proposed. Oneobservation in particular has led to the characterisation of numerousGAG-ligand models; the grouping of sulfation into discrete regions, or“domains”, along the length of many types of GAGs, such as heparansulfate. Interestingly no template for this phenomenon has yet beenobserved, and it appears to be primarily a result of the temporalactivity of the sulfotransferase enzymes responsible for this phase ofGAG synthesis.

Particularly useful tools in the study of specific GAG sequences are anumber of heparin lyases that can be used to examine targeteddepolymerisation of complex carbohydrate chains, thereby providinginsight into their structure. One particular heparan lyase, heparinaseIII (heparitinase), cleaves heparin sulfate chains at sites flanking thehighly sulfated domains that may occur in heparan sulfate chains. Thus,using this enzyme, it is possible to liberate these potentially activeregions from the full length sugar chains and separate them, if theyfunction as single domains, via affinity chromatography, from regionswith no specific affinity for the BMP2-HBP.

It is important to note that, in the case of GAG-ligand interactions,affinity by sequence does not necessarily guarantee bioactivity. Themode of activity mediated by GAGs during their association with theirvarious ligands differs greatly depending on the system. In someinstances where the sugar chain is responsible for prolongingprotein-protein interaction via stabilisation of tertiary proteinstructures, such as is found between FGF and its receptor, and theinteraction between HGF/SF and Met, multiple discrete sulfation regionsmay be involved in mediating the intended bioactivity of the sugarchain. In such instances the isolation of individual sulfated domainsfrom a full length carbohydrate chain may, in fact, result in aninhibition of sugar bioactivity since though each “domain-fragment”still binds its intended target it is unable to mediate the intendedbiological effect of a combined full length carbohydrate chain.

Interestingly, this particular characteristic of GAG-ligand interactionsis precisely what makes this manner of approach useful for modulatingBMP2 activity. The proposed model for GAG modulation of BMP2 bioactivityinvolves immobilization of the cytokine to GAGs in the ECM or on thecell surfaces. In this type of system the application of exogenous GAGsspecific to the heparin-binding domain of BMP2 would prevent thisinteraction, increasing short term BMP2 mediated signalling, similar tothe effect observed during the addition of soluble heparin. While thereis some indication that this manner of interaction would continue toprotect the cytokine from proteolytic degradation, delocalization ofBMP2 from its intended region of bioactivity has the potential tonegatively impact the cytokines effectiveness in the long term.

Control testing of our full length GAG+ and GAG− chains resulted insimilar profiles to those observed during their primary separation.Analysis of GAG+ and GAG− chains post treatment with heparinase III,however, gave surprising results. The digestion of GAG+ chains did notseem to generate separable fragments based on simple affinity for theBMP2-HBP. Furthermore, the digestion of full length GAG− chains yieldedno liberation of positive domains from the negative sugar chains. Thereis some possibility that the enzymatic digestion did not go tocompletion. However, the resulting chromatogram clearly showed a largeincrease in the absorbance at 232 nm when compared to the full lengthGAG chains. As a large proportion of the absorbance ofglycosaminoglycans at 232 nm is mediated via absorbance of unsaturatedbonds, such as those formed during enzymatic depolymerisation, itstrongly indicates that the enzymatic digestion was, in fact,successful.

The implications of this result are somewhat unusual. This data suggeststhat GAG chains are not only synthesised by cells to specificallyinteract with BMP2, but that, in the case of MC3T3 cells, these sugarchains carry a number of sequence repeats specific for aspects of BMP2metabolism. The fact that BMP2 is an extremely potent factor may offeran explanation for this observation. The effects of BMP2 on theosteoinduction of mesenchymal progenitor cells is well documented, as isits ability to induce ectopic bone formation in cells that are even moreremoved from the osteogenic lineage. Given this potency, aberrantsignalling of BMP2 is known to have deleterious consequences both forhealing and in development. It is possible that numerous repeats of theBMP2-HBP interaction sequence on preosteoblast GAGs are designed toensure a maximal binding, and thereby the modulation, of this cytokine'sability to induce altered cell fate. Conversely, the extremely lowconcentrations of BMP2 produced in vivo may also require this type ofsugar chain production in order to ensure the retention of a sufficientlocal concentration, an observation supported by the extremely highconcentrations of BMP2 required in vitro to induce the osteogenicdifferentiation of C2C12 myoblast cells.

Of particular interest is the fact that this repetition of BMP2-bindingdomains is produced via a synthesis pathway for which no template ortiming mechanism has yet been elucidated. The accuracy andreproducibility of sequence specific domains within a single sugar chain(as opposed to the random clustering of such domains with those againstother ligands) strongly suggests that these cells do, in fact, have theability to direct the generation of specific sugar sequences. Thecurrent understanding of HS structure implicates the progressivepost-synthesis “editing” of the carbohydrate chain in the generation ofsequence-specific regions, with observations pointing towards somemanner of enzymatic “template”, whereby the local concentrations ofparticular sulfotransferases as well as other interacting molecules areused to directly control the generation of specific sugar sequences. Ourcurrent understanding of this mode of specific synthesis is largelyformulated based on numerous studies including those by Lindahl et al.that investigated the high affinity interaction between antithrombin IIIand heparin, and those by Esko et al. involving Chinese hamster ovary(CHO) cell mutants with altered GAG synthesis pathways. These studies,while varying significantly in their approaches to GAG analysis, allpoint towards a highly conserved system of specific GAG synthesis, forthe directed modulation of cytokine and receptor activity. Importantly,these studies also serve to explain the potential generation of suchBMP2 repeats as were observed in our study.

GAG+ Constitution Full Length GAG+ Size

The bioactivity of individual GAGs chains for FGFs is closely related tocarbohydrate chain length. A common approach to assessing GAGbioactivity is to assay ever shorter sulfated domain fragments and sodetermine the shortest possible sequence required to mediate theactivity observed.

Using this approach we first examined full length GAG+ sugar chains, anddetermined that they were >20 KDa in size, long enough to carry multipledomains with affinity for BMP2. Interestingly, this observation providedsupport for the earlier observation that GAG+ samples treated withheparinase 3 showed multiple repeats of carbohydrate chain segments witha specific affinity for BMP2, since a variably sulfated sugar chain ofthis size has the capacity to carry numerous sulfated domains.

GAG+ Sugar Species

With the majority of the five glycosaminoglycan types that constitutethe “glycome” able to encode the observed specific interactions withBMP2, it was necessary to elucidate which of these GAG types could beinvolved in this specific association. Although the prime candidate forthis interaction is a heparan sulfate, analogous growth factorinteractions have also been identified for chondroitin and dermatansulfates.

Heparan sulfate can be totally depolymerised into its disaccharidecomponents with nitrous acid. This particular characteristic, sharedwith heparin and keratan sulfate, is essential for the analysis ofspecific GAG populations. In the case of our analysis of thecarbohydrate constituents of our GAG+ samples, degradation due tonitrous acid was diagnostic of heparan sulfate. This probability isprimarily due to its heparan sulfate's higher degree of chargepatterning via sulfation in comparison to either heparin or keratansulfate. Ultimately, this charge patterning is responsible for BMP2'sspecific interaction with HS.

Our analysis utilising the nitrous acid protocol showed a completedegradation of the GAG+ sample set indicating that the majority ofsugars in the GAG+ sample set were in fact 1,3-linked and, thus, wereheparan sulfate. This result supports the numerous observations inregards to the specificity of heparan sulfate cytokine interactions,particularly the interaction that BMP2 exhibits with heparin and HS.

GAG Species Analysis BMP2-HBP Specific GAGs (Alternative Species)

The small remnant peak that was observed after the degradation of GAG+samples by nitrous acid supports the possibility that other sulfatedGAGs carrying some specific affinity for BMP2 may be found in the GAG+sample set. Given our current understanding of the role of sulfation inmediating the interaction between GAGs and BMP2, chondroitins anddermatans are the most likely alternative sugars to show a specificinteraction with BMP2 as these show the highest potential diversity insulfation patterns.

A methodology frequently employed for GAG analysis includes examiningthe role of individual sulfation positions on GAG-ligand interactions.This method of analysis gives an indication of the importance ofindividual sulfation positions in maintaining the interaction betweenthe GAG chain and its specific target. Furthermore, since the differentspecies of GAGs only have the potential to carry sulfation patternsspecific to their species, this can aid in narrowing the possibleglycosaminoglycan candidates that may show an affinity for a specificligand.

To this end we examined the affinity for the BMP2-HBP carried byvariably sulfated CS chains, C4S and C6S, and standard DS.Interestingly, only C4S carried any significant affinity for theBMP2-HBP. This data indicates that it is likely that the 4-O-sulfationis necessary for CS to interact with the BMP2-HBP. Interestingly,dermatan sulfate showed no affinity for the BMP2-HBP. This observationis of interest since DS is the only CS species that demonstratesdiversity in sulfation similar to that of HS. Furthermore, ourobservations indicate a possibility that the epimerisation of GlcA toIdoA in DS compromises the ability of this sugar type to bind theBMP2-HBP. Both C4S and DS are able to carry 4-O-sulfation, yet onlysmall quantities of DS were retained on the column in comparison to C4S.Alternatively, this lack of affinity may simply be due to thisparticular batch of DS not carrying sufficient 4-O-sulfation toeffectively mediate binding to the BMP2-HBP. Interestingly, theseparticular observations appear to demonstrate an interaction betweenBMP2 and CS carrying 4-O-sulfation. While previous studies haveinvestigated the use of CS-BMP2 interactions in drug delivery systems,not much is known about any sequence specific interaction betweenindividual CS species and BMP2. However, since HS chains are composed of1,4-linked disaccharide units, the observed 4-O-sulfation responsiblefor CS-BMP2 interactions is not found in HS-BMP2 interactions, pointingto a sequence specific interaction not found in CS. Thus it is likelythat the remnant peak observed post-nitrous acid treatment may containsmall quantities of 4-O-sulfate carrying C4S or DS.

Further investigation revealed that neither commercial HS nor heparinheld any significant affinity for the peptide column. The HS used forthis assay was purchased commercially from Sigma-Aldrich and was derivedfrom bovine kidney. Given what is known about the tissue specificity ofHS it is possible that this commercially available HS, isolated frombovine kidney sources, carried negligible carbohydrate sequencesrequired to specifically mediate an interaction with BMP2. Similarlyneither LMW nor HMW heparin showed any affinity for the peptide column.The heparin used for this analysis was also purchased fromSigma-Aldrich, and was derived from porcine intestinal mucosa.

While heparin's interaction with antithrombin III has been wellcharacterised, and notwithstanding its versatile role in the isolationof susceptible molecules, heparin's interaction with growth factors isnot, in general, regarded to be specific due to its uniform sulfation.However, given that heparin is routinely used to isolate BMP2, it issomewhat surprising that neither of the heparin samples interacted withthe peptide column to any significant degree.

A further possibility for this lack of interaction between the peptidecolumn and heparin is due to the difference in molecular weights betweenthe two molecules. The small BMP2-HBP attached to the column may havedifficulty in maintaining its association with the larger, heavilysulfated heparin chain. The inability of heparinase-cleaved heparin tobind the column, however, appeared to indicate that the steric effectsof using full length heparin on the column were not solely responsiblefor disrupting the potential interaction between the sugars and theBMP2-HBP. There is no immediately apparent reason for this inability forcommercial heparin to associate with the BMP2-HBP column, though it maybe postulated that further spatial separation of the BMP2-HBP from itsassociated bead via spacer chains may help to ameliorate this problem.

SUMMARY

In this study we have demonstrated the use of affinity chromatography toisolate a subset of glycosaminoglycans that carry a specific affinityfor the BMP2-HBP, and have shown the potential for this procedure toyield reproducible results. During this portion of our investigationinto the interaction between matrix based GAGs and BMP2, we have madeseveral observations with regards to both the type of GAGs involved inmediating this association and their structure.

Our results have implicated heparan sulfate for mediating the majorityof the affinity BMP2 has for the preosteoblast ECM, an interaction whichis increasingly recognised as being responsible for the modulation ofBMP2 activity. Furthermore, our investigation into the likely structureof the ECM-resident GAGs isolated on the basis of their affinity for theBMP2 heparin-binding site have yielded a surprising result.

Our data indicates that full length BMP2 GAG+ chains do not consist ofindividual domains with specific affinity for BMP2 interspersed withregions of little or no affinity for the factor. Instead, our resultsimply that these GAG+ chains consist of multiple BMP2-binding domainrepeats. This result is surprising on several levels. Firstly, therepetition required to fulfil this observation over the full length ofa >20 kDa carbohydrate chain points to the presence of some manner ofsynthetic template. Indeed, while previous studies have been unable toderive a template for the assembly of tissue-specific GAG chains, thevery fact that such specificity exists supports the presence of atemplate-based system. Although no genomic template has been elucidatedfor this process there exists some possibility of a proteomic, perhapsenzymatic, template.

Secondly, this observation provides some evidence as to the importanceof the interaction between BMP2 and GAGs. Multiple repeats of the BMP2affinity site along the length of the carbohydrate chain may be requiredto ensure maximal binding of BMP2 to the ECM. This particularassociation has been shown to significantly lengthen the factor's halflife, as well as probably being responsible for maintaining asignificant local concentration in order to maintain signalling.Alternatively, some studies have proposed a model whereby BMP2 isspatially inhibited from interacting with its receptors due to theinteractions with ECM-based GAGs. In this particular scenario therepetition of BMP2 affinity sequences would ensure a maximal binding ofthe factor, thus reducing the chance of it interacting with itsreceptors.

Our cumulative results indicated that this system for the isolation ofGAGs from the ECM is viable and likely to yield GAG chains that have aspecific affinity for BMP2.

This study supports previous findings in regards to the interactionbetween GAGs and BMP2. Although the prevention of BMP2 associating withthe ECM in vitro through the addition of exogenous GAG+ appears toincrease BMP2 signalling and upregulates osteogenic gene expression,observations to the contrary have also reported. In these studies, invivo examination of BMP2's modulation via the HBP showed a distinctimprovement in long term osteogenesis when the association with ECM GAGswas increased. It is possible that this interaction plays a major rolein maintaining local concentrations by preventing the factor fromdiffusing away from its sites of primary activity. In light of thesestudies and our own observations, we propose that BMP2's activity isboth positively and negatively regulated by its association with GAGs.Negative regulation may occur precisely via the model proposed byKatagiri and colleagues, whereby the retention of BMP2 in the ECM, awayfrom its receptors, leads to a downregulation of BMP2 signalling.However, cells that require signalling by this factor may potentiallysecrete various enzymes to remodel extracellular sugar chains, such assulfatases and heparinases, in order to “clip away” GAGs retaining BMP2in the ECM, thereby liberating the factor and allowing it to signal,leading to the BMP2-ECM interaction ultimately becoming one of positivemaintenance of the cytokine's activity. Alternatively, negativeregulation of BMP2 by cell surface GAGs, may be via the internalisationof GAG chains with their associated BMP2 molecules, as has been observedby Jiao and colleagues.

These previous studies, in conjunction with our own observations, havelead us to conclude that the sequence-specific interplay between BMP2and heparin sulfate represents an intricate control mechanism that hasthe capacity to both positively and negatively regulate BMP2 signaling.Physiologically this interaction is responsible for enforcing contextdependent responses to this potent cytokine in respect to many facets ofembryonic development, precursor commitment and wound healing.

Example 2 Purification of BMP2 Peptide Specific HS

We used a peptide having heparin-binding properties from the matureBMP-2 sequence to identify novel HS that bind to the peptide.

Mature BMP-2 amino acid sequence: [SEQ ID NO: 14]QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVL KNYQDMVVEGCGCRHeparin-binding peptide amino acid sequence: [SEQ ID NO: 1]QAKHKQRKRLKSSCKRHP

To replicate the natural presentation of the heparin-binding site webiotinylated the peptide on it's C-terminus and kept the proline (P) toimprove the flexibility/accessibility of the peptide once bound to thestreptavidin column.

Isolation of BMP2 Peptide Specific HS

Materials used included a BMP2—peptide coupled Streptavidin column,HiPrep Desalting Column (GE Healthcare), 20 mM PBS+150 mM NaCl (Low SaltBuffer), 20 mM PBS+ 1.5 M NaCl (High Salt Buffer), HPLC grade Water(Sigma), Biologic-Duoflow Chromatography system (Bio-Rad) and a FreezeDrier.

The column was equilibrated with Low Salt buffer and 1 mg SigmaHS(H9902) was dissolved in low salt buffer and passed through theBMP2-Streptavidin column. Unbound media components were removed from thecolumn by washing low salt buffer (20 mM PBS, pH 7.2, 150 mM NaCl) untilthe absorbance of the effluent at 232 nm almost return to zero. HS boundto the matrix was eluted with high salt buffer (20 mM PBS, pH 7.2, 1.5 MNaCl). Peak fractions were pooled and freeze dried for 48 hrs.

HS 1 mg was applied to the column and washed with 20 mM PBS buffercontaining a low (150 mM) NaCl concentration. After washing with lowsalt buffer, the bound HS were eluted with 20 mM PBS buffer containing ahigh (1.5 M) NaCl concentration. Peaks representing retained fractions(monitored at 232 nm) were collected and subjected to further desalting.

After freeze drying 6 mg of positive HS (GAG+) and 1.8 mg of negative HS(GAG−) were obtained.

Example 3 Evaluation of BMP-2 Specific Heparan Sulfates

C2C12 are mouse mesenchymal stem cells normally exhibiting myogenicdifferentiation but capable of being directed in the osteogenic lineagewith supplementation of BMP-2 at passage 3. C2C12 cells at passage 3were maintained in DMEM with 1000 g/L glucose (low glucose), 10% of FCS,1% of P/S and without L-glutamine (maintenance media).

DMEM with 1000 g/L glucose (low glucose), 5% of FCS, 1% of P/S andwithout L-glutamine was used as differentiation media.

Effect of BMP-2 on Osteogenesis

We evaluated the effects of exogenous BMP-2 on osteogenesis by measuringthe levels of expression of osteogenic markers (osteocalcin, osterix,Runx2).

Through assaying the effect of addition of different amounts (100 ng/mland 300 ng/ml) of BMP-2 to the cells we observed a significant decreaseat day 5 in the expression of osterix, osteocalcin and Runx2 in cellshaving 100 ng/ml BMP-2 compared to addition of 300 ng/ml BMP-2 (FIGS.27-29). Thus we chose this time point for future tests, as any changesshould be readily observable.

Materials and Methods

C2C12 cells at passage 3 were used. Cells were kept in liquid Nitrogenat Passage 3 with 1×10⁶ cells/vial. Once cells were taken from liquidNitrogen, we added 500 μl of culture media, pipetted up and down torefreeze the cells and immediately added 15 ml of culture media.

Culture media was DMEM with 1000 g/L glucose (low glucose), 10% of FCS,1% of P/S and without L-glutamine. Treatment media was DMEM with 1000g/L glucose (low glucose), 5% of FCS, 1% of P/S and without L-glutamine.

C2C12 cells were allowed to grow to 75% confluence before harvesting(normally 2 to 3 days) in culture media.

Cells were counted as follows. Media was first aspirated/discarded; 15ml of PBS added, discard the PBS and add 3 ml of trypsin, incubate at37° C. for 5 min to lift the cells from the flask. 9 ml of culture mediaadded to neutralize the trypsin. GUAVA used to determine the amount ofcells for subsequent cell seeding onto the experiment plates. Forexample, for 3 sets of 12-well plates 30,000 cells×36 wells×3.7cm²=4,000,000 cells. Dilute the cells from the stock and add the desiredamount of culture media for cell seeding (each well requiring 500 μl ofmedia with 30,000 cells).

To prepare BMP2 stock 10 μg rhBMP2 (Bone Morphogenetic Protein 2) wasre-suspended in 100 μl of 4 mM HCl/0.1% BSA.

The following RNA extraction protocol was used. 350 μl of RA1 buffer wasused for cell lysis. Cells were frozen with RA1 at −80° C. for one dayafter which cells were thawed and the lysate filtered for 1 min at11,000 g. The filtrate was mixed with 350 μl 70% ethanol in 1.5 ml tubesand centrifuged for 30 s at 11,000 g. 350 μl of MDB buffer was added andthe mixture centrifuged for 1 min at 11,000 g. 95 μl of Dnase reactionmixture added and mixture left at room temperature for at least 15 min.Then wash with 200 μl of RA2 buffer (to deactivate the Dnase), andcentrifuge for 30 s at 11,000 g. Wash with 600 μl of RA3 buffer,centrifuge for 30 second at 11,000 g. Wash with 250 μl of RA3 buffer,centrifuge for 2 min at 11,000 g. Elute the RNA with 60 μl of Rnase-freeH₂O, centrifuge for 1 min at 11,000 g. Measure the concentration usingNanodrop (unit in ng/μl).

RT (reverse-transcription) experiments were performed as follows. Thefollowing were mixed in a PCR tube: Random Primer (0.1 μl), DNTP (1 μl),RNA (250/500 ng), Rnase-Free H₂O (topped up to a final volume of 13 μl).Incubate at 65° C. for 5 min. Incubate on ice for at least 1 min.Collect the contents and centrifuge briefly before adding: 1^(st) StrandBuffer (4 μl), DTT (1 μl), RnaseOUT (1 μl), SSIII Reverse (1 μl). Top upto final volume of 20 μl. Mix by pipetting up and down. Incubate at roomtemperature for 5 min. Incubate at 50° C. for 60 mins. Inactivate thereaction at 70° C. for 15 min.

Reverse-transcription experiments were performed twice on separate daysand the PCR products pooled together and diluted to a finalconcentration of 2.5 ng/μl for subsequent Real-Time PCR.

The Real-Time PCR was performed using a TaqMan® Fast Universal PCRmaster Mix (2×) (Applied Biosystem). PCR master Mix (10 μl), ABI probe(1 μl), cDNA (1 μl), ddH₂O (8 μl). GAPDH and Beta actin were used ascontrol genes against the experimental targets OSX (osterix), OCN(Osteocalcin) and Runx2.

Effect of BMP-2 Specific HS GAG+ on Osteogenesis

We evaluated the effects of the BMP-2 specific HS (GAG+) isolated inExample 2 on osteogenesis by measuring the levels of expression ofosteogenic markers (osterix, Runx2, alkaline phosphatase and BspII) byquantitative polymerase chain reaction (qPCR). A time course wasprepared to compare the expression of the markers over a course of 10days to compare the control to a low and a high dose of BMP-2, the highdose being the optimal conditions to induce differentiation of thecells.

Materials and Methods

Cells were seeded at 30,000 cell/cm² in maintenance media and left toattach overnight. The following day we switched to differentiation mediawith:

-   -   No additives    -   100 ng/ml BMP-2 (positive control)    -   100 ng/ml BMP-2+30 μg/ml−GAG (Neg GAGs)    -   100 ng/ml BMP-2+30 μg/ml+GAG (Pos GAGs)    -   100 ng/ml BMP-2+30 μg/ml Heparin (Sigma # H3149)    -   100 ng/ml BMP-2+30 μg/ml Total Heparan Sulfate (Sigma # H9902—HS        prior to fractionation)

The carbohydrates and BMP-2 were mixed together in the smallest volumepossible and incubated at room temperature for 30 minutes before theiraddition to the media and on the cells.

After 5 days, RNA was extracted using the Macherey-Nagel kits andReverse-Transcription was performed.

As we show in FIGS. 30-33, the Heparan sulfate from porcine mucosa(Total HS) can increase the activity of BMP-2 (shown through GAG+induced increases in the expression of Alkaline Phosphatase, osterix,BspII and Runx2) and this activity is contained within the fraction thatbinds BMP2 (Pos GAGs). This means that we can isolate the BMP enhancingfraction of a commercial HS by passing them on the BMP-HBD peptidecolumn.

Example 4

MC3T3-E1 (s14) preosteoblast cells (a mouse embryo calvaria fibroblastcell line established from the calvaria of an embryo) were expanded inαMEM media supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodiumpyruvate and Penicillin/Streptomycin every 72 hours until sufficientcells were generated for plating. The cells were differentiated byplating at 5×10⁴ cells/cm² in αMEM media supplemented with 10% FCS, 2 mML-glutamine, 25 μg/ml ascorbic acid, 10 mM β-glycerol phosphate andPenicillin/Streptomycin. The media was changed every 72 hours for 8 daysat which point the cells and media were harvested. The media wasretained and clarified by high speed centrifugation and filtrationthrough a 0.4 μm filter. The cell layer was disrupted using a cellscraper and an extraction buffer containing PBS (150 mM NaCl w/o Ca²⁺and Mg²⁺), 1% CHAPS, 8 M Urea and 0.02% NaN₃.

At all stages (unless otherwise stated), samples were clarified beforeloading onto column systems. This process included high speedcentrifugation at 5000 g for 30 min, and filtration through a 0.4 μmsyringe filter. The samples were always clarified directly prior toloading through the column system to prevent precipitates forming instagnant solutions.

Anion exchange chromatography was used to isolateproteoglycosaminoglycan (PGAG) fractions from both the media and celllayer samples. In each case, the media or cell layer samples were runthrough a Pharmacia XK 26 (56-1053-34) column packed with Capto Q AnionExchange Beads (Biorad) at a flow rate of 5 ml/min on a Biologic DuoFlowsystem (Biorad) using a QuadTec UV-Vis detector. The samples were loadedin a low salt buffer containing PBS (150 mM NaCl w/o Ca²⁺ and Mg²⁺), 100mM NaCl, 0.02% NaN₃ at pH 7.3. The samples were eluted in a high saltbuffer containing PBS (150 mM NaCl w/o Ca²⁺ and Mg²⁺), 850 mM NaCl and0.02% NaN₃ at pH 7.3. The relevant fractions were collected and pooledinto a single PGAG sample and lyophilized in preparation for desalting.

The PGAG sample was desalted through four sequentially joined PharmaciaHiPrep™26/10 (17-5087-01) columns at a flow rate of 10 ml/min on aBiologic DuoFlow system (Biorad) using a QuadTec UV-Vis detector. Therelevant fractions were collected and pooled into a single sample setand lyophilized in preparation for further treatment.

In the fourth step, the PGAG sample set obtained from the desaltingprocedure was subjected to a pronase and neuraminidase treatment, inorder to digest away core proteins and to subsequently liberate GAGchains. In this respect, lyophilized PGAG samples were resuspended in aminimum volume of 25 mM sodium acetate (pH 5.0) and clarified byfiltration through a 0.4 μm syringe filter. The total sample volume wasdispensed into 10 ml glass tubes in 500 μl aliquots. To this aliquot wasadded 500 μl of 1 mg/ml neuraminidase before the mixture was incubatedfor 4 hours at 37° C. Following incubation, 5 ml of 100 mM Tris-acetate(pH 8.0) was added to each sample. An additional 1.2 ml of 10 mg/mlpronase, reconstituted in 500 mM Tris-acetate and 50 mM calcium acetate(pH 8.0), was added to each sample before the mixture was incubated for24 hrs at 36° C. Following this treatment, all volumes were combined andprepared for anion exchange chromatography by centrifugation andfiltration.

In a fifth step, the GAG sample isolated following protein cleavage waseluted through a Pharmacia XK 26 (56-1053-34) column packed with Capto QAnion Exchange Beads (Biorad) at a flow rate of 5 ml/min on a BiologicDuoFlow system (Biorad) using a QuadTec UV-Vis detector. In thisrespect, the sample was loaded in a low salt buffer containing PBS (150mM NaCl w/o Ca²⁺ and Mg²⁺) and 0.02% NaN₃ at pH 7.3. The sample waseluted in a high salt buffer containing PBS (150 mM NaCl w/o Ca²⁺ andMg²⁺), 850 mM NaCl and 0.02% NaN₃ at pH 7.3. The relevant fractions werepooled, lyophilized and desalted as per the aforementioned protocol fordesalting the PGAG sample.

N-terminal biotinylated peptide (1 mg), corresponding to theheparin-binding domain of BMP-2, and comprising an amino acid sequencerepresented by QAKHKQRKRLKSSCKRH [SEQ ID NO: 17], was mixed with lowsalt buffer containing PBS (150 mM NaCl w/o Ca²⁺ and Mg²⁺). The mixturewas eluted through a column packed with a streptavidin-coated resinmatrix. The column was then exposed to a high salt buffer containing PBS(150 mM NaCl w/o Ca²⁺ and Mg²⁺), 850 mM NaCl and 0.02% NaN₃ at pH 7.3,to ascertain whether, under those conditions the peptide had boundsecurely to the matrix. No substantial loss of peptide from the columnwas observed. The column was subsequently washed with the low saltbuffer in preparation for sample loading.

The GAG mixture (2 mg), isolated using the procedure outlined in Example1, was suspended in low salt sodium phosphate buffer (1 mL), and loadedonto the peptide column of Example 2. The sample was eluted with a lowsalt buffer containing PBS (150 mM NaCl w/o Ca²⁺ and Mg²⁺). A peakcorresponding to GAGs with negligible BMP-2 affinity was observed in theUV-Vis detector trace. The column fractions responsible for giving riseto this peak were combined. These fractions are known as ‘GAG−’—theminus sign denoting the lack of affinity with the column. When it becameevident from the UV-Vis detector that the trace had flattened to thebaseline, and that no further oligosaccharide was eluting, the elutingsolvent was changed to a high salt buffer containing PBS (150 mM NaClw/o Ca²⁺ and Mg²⁺), 850 mM NaCl and 0.02% NaN₃ at pH 7.3. Following thischange in the eluting solvent, a peak corresponding to BMP-2 specificGAGs was observed in the UV-Vis detector trace. The column fractionsresponsible for giving rise to this peak were combined. These fractionsare known as ‘GAG+’—the plus sign denoting the presence of affinity withthe column. In the case of GAG compounds sourced from preosteoblastcells, the GAG+ fraction represented 10% of the overall GAG mixture.

Example 5

The addition of BMP2 has a clearly defined capacity to induce osteogenicdifferentiation in C2C12 myoblasts. Similarly, the pre-incubation ofBMP2 with heparin has been shown to both extend the cytokines half lifeand its immediate potency in vitro. Here we examined the capacity ofGAG+ and GAG− fractions to augment the osteoinduction of C2C12 cells invitro by BMP2.

The GAG+ sample from Example 4 (0, 10, 100, 1000 ng/mL) was added toC2Cl2 myoblasts in vitro in the presence of BMP-2 (0, 50, 100 ng/mL).Measurement of the relative expression of the osteocalcin gene indicatedthat the GAG+ sample was able to potentiate BMP-2 to effect osteocalcingene expression at levels of BMP-2 far below those currently used intherapy (300 ng/mL). The results of this assay (including calculatedp-values and errors) are represented graphically in FIG. 34 in which theexperimental conditions for each ‘culture condition’ are as follows:

-   -   1. Control cells, no BMP-2 added, no GAG added    -   2. BMP-2 at 50 ng/mL    -   3. BMP-2 at 50 ng/mL, GAG+ at 10 ng/mL    -   4. BMP-2 at 50 ng/mL, GAG+ at 100 ng/mL    -   5. BMP-2 at 50 ng/mL, GAG+ at 1000 ng/mL    -   6. BMP-2 at 100 ng/mL    -   7. BMP-2 at 100 ng/mL, GAG+ at 10 ng/mL    -   8. BMP-2 at 100 ng/mL, GAG+ at 100 ng/mL    -   9. BMP-2 at 100 ng/mL, GAG+ at 1000 ng/mL

Interestingly, while 1000 ng/ml of GAG+ is able to significantly augmentBMP2 mediated osteocalcin expression, the addition of concentrations ofGAG+ below 1000 ng/ml appear to progressively inhibit this expression.Furthermore, the addition of sufficient GAG+ also managed to drive theinduction of osteocalcin by 50 ng/ml of BMP2 above that of 100 ng/ml ofBMP2 on its own, indicating the potency of this interaction.

This cell culture based analysis demonstrated that the addition of GAG+to C2C12 osteogenic cultures together with BMP2 resulted in asignificant upregulation of osteocalcin expression indicating anincrease in BMP2 signalling efficacy. This result supports the specificassociation of GAG+ chains with BMP2, thereby blocking the BMP2-HBP andpreventing its association with matrix-based PGAGs. The resultingupregulation of osteogenic gene expression is comparable to thatobserved in previous studies utilising heparin to achieve a similareffect. Interestingly, the addition of concentrations of GAG+ that fallbelow 1000 ng/ml appear to have an initially antagonistic effect on BMP2signalling.

One possible hypothesis to explain this observation revolves around thecapacity for a given number of GAG+ molecules to bind a certain numberof BMP2 molecules. Under conditions where no exogenous GAG+ is added tothe culture system the majority of BMP2 molecules will be able toassociate with the ECM, thereby being localised away from their cognatereceptors and being unable to immediately initiate signalling.Subsequent dissociation of BMP2 from the ECM, both spontaneously and bytargeted enzymatic alteration of their associated GAG chains, has thecapacity to induce long term BMP2 signalling. The addition of a largenumber of GAG+ molecules to this system, as is the case in samplessupplemented with 1000 ng/ml of GAG+, permits the majority of BMP2molecules to remain in solution where they are free to mediate receptordimerisation and induce downstream signalling. Both these processes ofcytokine/receptor interaction likely require particular concentrationthresholds in order maintain an efficient level of signalling. Underculture conditions containing 50 ng/ml of BMP2, the addition of lowconcentrations of GAG+ allows for a portion of the available cytokine toremain soluble while the remaining portion associates with the ECM.Under these conditions only a small quantity of BMP2 remains solublebut, due to its low concentration, becomes highly diffuse in the medialeading to negligible signalling. Similarly, due to a portion of theBMP2 remaining solubilised, a reduced quantity of BMP2 can be found inthe ECM, resulting in a decrease in signalling from BMP2 liberated fromthe ECM by direct cellular activity. However, under culture conditionscontaining 100 ng/ml of BMP2 the combined effects of soluble and ECMbased BMP2 are, with the addition of 100 ng/ml of GAG+, sufficient toinduce BMP2 signalling similar to control levels. Without further study,however, the dynamics involved in BMP2/GAG+ signalling remain unclear.Future studies utilising surface plasmon resonance may help elucidatethe efficiency of BMP2/GAG+ interactions and may aid in clarifying theseobservations.

Example 6

The enzyme heparanase 3 was used to cleave GAG+ and GAG− sugar chainsfrom Example 4 according to the following method. GAG+ and GAG− wereeach treated separately at a concentration of 4 mg/mL, with heparanase 3(250 mU enzyme per 100 μg oligosaccharide) for 16 hours at 37° C.Subsequently, the mixture was heated for 5 minutes at 70° C. toinactivate the heparanase 3. The digested GAG+ and GAG− mixtures wereeach subjected to the peptide column separately. The UV-Vis detectortrace of each chromatographic run indicated that the digested materialshowed the same affinity for the column as the undigested material.

1. A method of isolating glycosaminoglycans capable of binding to aprotein having a heparin-binding domain, the method comprising: (i)providing a solid support having polypeptide molecules adhered to thesupport, wherein the polypeptide comprises a heparin-binding domain;(ii) contacting the polypeptide molecules with a mixture comprisingglycosaminoglycans such that polypeptide-glycosaminoglycan complexes areallowed to form; (iii) partitioning polypeptide-glycosaminoglycancomplexes from the remainder of the mixture; (iv) dissociatingglycosaminoglycans from the polypeptide-glycosaminoglycan complexes; (v)collecting the dissociated glycosaminoglycans.
 2. A method ofidentifying glycosaminoglycans capable of stimulating or inhibiting thegrowth and/or differentiation of cells and/or tissues, the methodcomprising: (i) providing a solid support having polypeptide moleculesadhered to the support, wherein the polypeptide comprises aheparin-binding domain; (ii) contacting the polypeptide molecules with amixture comprising glycosaminoglycans such thatpolypeptide-glycosaminoglycan complexes are allowed to form; (iii)partitioning polypeptide-glycosaminoglycan complexes from the remainderof the mixture; (iv) dissociating glycosaminoglycans from thepolypeptide-glycosaminoglycan complexes; (v) collecting the dissociatedglycosaminoglycans; (vi) adding the collected glycosaminoglycans tocells or tissues in which a protein containing the amino acid sequenceof the heparin-binding domain is present; (vii) measuring one or moreof: proliferation of the cells, differentiation of the cells, expressionof one or more protein markers.
 3. The method of claim 1 or 2 whereinthe mixture comprising glycosaminoglycans contains extracellular matrixmaterial.
 4. The method of claim 3 wherein the extracellular matrixmaterial is derived from connective tissue or connective tissue cells.5. The method of any one of claims 1 to 4 wherein the mixture comprisingglycosaminoglycans contains one or more of a dextran sulphate, achondroitin sulphate, a heparan sulphate.
 6. The method of any one ofclaims 1 to 5 wherein the mixture comprising glycosaminoglycans has beenenriched for one of dextran sulphate, chondroitin sulphate, heparansulphate.
 7. The method of any one of claims 1 to 6 wherein the methodfurther comprises subjecting the collected glycosaminoglycans to furtheranalysis in order to determine structural characteristics of the GAG. 8.The method of any one of claims 1 to 7 wherein theglycosaminoglycan-polypeptide complexes are contacted with a lyase. 9.The method of any one of claims 1 to 8 wherein the polypeptide is, orcomprises, one of SEQ ID NO.s: 1-13 or
 17. 10. Heparan sulphate HS/BMP2.11. Culture media comprising HS/BMP2.
 12. A pharmaceutical compositionor medicament comprising HS/BMP2.
 13. The pharmaceutical composition ormedicament of claim 12 further comprising a pharmaceutically acceptablecarrier, adjuvant or diluent.
 14. The pharmaceutical composition ormedicament of claim 12 or 13 further comprising BMP2 protein.
 15. Apharmaceutical compositions or medicament according to any one of claims12 to 14 for use in the prevention or treatment of injury or disease.16. Use of HS/BMP2 in the manufacture of a medicament for the preventionor treatment of injury or disease.
 17. The pharmaceutical composition oruse of claim 15 or 16 wherein the prevention or treatment is chosenfrom: repair, regeneration or replacement of connective tissue and woundhealing.
 18. A method of preventing or treating injury or disease in apatient in need of treatment thereof, the method comprisingadministering an effective amount of HS/BMP2 to the patient.
 19. Themethod of claim 18 wherein the administered HS/BMP2 is formulated as apharmaceutical composition or medicament.
 20. The method of claim 19wherein the pharmaceutical composition or medicament further comprisesBMP2 protein.
 21. A method of promoting or inhibiting osteogenesiscomprising administering HS/BMP2 to bone precursor cells or bone stemcells.
 22. The method of claim 21 wherein the bone precursor cells orbone stem cells are contacted with HS/BMP2 in vitro.
 23. The method ofclaim 21 wherein the bone precursor cells or bone stem cells arecontacted with HS/BMP2 in vivo.
 24. The method of any one of claims 21to 23 wherein the bone precursor cells or bone stem cells are contactedwith BMP2 simultaneously with HS/BMP2.
 25. A method of promoting orinhibiting the formation of cartilage tissue comprising administeringHS/BMP2 to cartilage precursor cells or cartilage stem cells.
 26. Themethod of claim 25 wherein the cartilage precursor cells or cartilagestem cells are contacted with HS/BMP2 in vitro.
 27. The method of claim25 wherein the cartilage precursor cells or cartilage stem cells arecontacted with HS/BMP2 in vivo.
 28. The method of any one of claims 25to 27 wherein the cartilage precursor cells or cartilage stem cells arecontacted with BMP2 simultaneously with HS/BMP2.
 29. A method for therepair, replacement or regeneration of connective tissue in a human oranimal patient in need of such treatment, the method comprising: (i)culturing mesenchymal stem cells in vitro in contact with HS/BMP2 for aperiod of time sufficient for said cells to form connective tissue; (ii)collecting said connective tissue; (iii) implanting said connectivetissue into the body of the patient at a site of injury or disease torepair, replace or regenerate connective tissue in the patient.
 30. Themethod of claim 29 further comprising contacting the mesenchymal cellsin culture with exogenous BMP2.
 31. Connective tissue obtained by invitro culture of mesenchymal stem cells in the presence of HS/BMP2. 32.Connective tissue as claimed in claim 31, wherein the mesenchymal cellsare cultured in the presence of exogenous BMP2, and optionally in thepresence of BMP2.
 33. A pharmaceutical composition comprising stem cellsand HS/BMP2.
 34. The pharmaceutical composition of claim 32 wherein thestem cells are mesenchymal stem cells.
 35. The pharmaceuticalcomposition of claim 33 or 34 wherein the composition further comprisesBMP2.
 36. A pharmaceutical composition according to any of claims 33 to35 for use in the treatment of injury or disease.
 37. A method for thetreatment of injury or disease in a patient in need of treatment thereofcomprising administering to the patient a pharmaceutical compositioncomprising stem cells and HS/BMP2.
 38. The method of claim 37 whereinthe stem cells are mesenchymal stem cells.
 39. The method of claim 37 or38 wherein the method further comprises administering BMP2 to thepatient.
 40. Use of HS/BMP2 in the growth of connective tissue in vitro.41. A method for growing connective tissue in vitro comprising culturingmesenchymal stem cells in contact with exogenously added HS/BMP2.
 42. Abiological implant comprising a solid or semi-solid matrix materialimpregnated with HS/BMP2.
 43. The biological implant of claim 42 furtherimpregnated with BMP2.
 44. The biological implant of claim 42 or 43further impregnated with mesenchymal stem cells.
 45. A kit comprising apredetermined amount of a glycosaminoglycan having high affinity for aprotein having a heparin-binding domain and a predetermined amount ofsaid protein.
 46. The kit of claim 45 wherein the glycosaminoglycan isHS/BMP2 and the protein is BMP2.